TY - JOUR
AU1 - Calenberg, Michael
AU2 - Brohsonn, Uwe
AU3 - Zedlacher, Marlies
AU4 - Kreimer, Georg
AB - Abstract Little is known about phototactic signal transduction in flagellate green algae; therefore, eyespot apparatuses, which are the light-sensitive “organelles” involved in photoorientation of these algae, were isolated and analyzed for the presence of heterotrimeric guanine nucleotide binding proteins (G proteins) and their coupling to the retinal-based photoreceptor. Specific high-affinity 35S-GTP-γ-S binding and GTPase activity, with sensitivity toward antibodies raised against vertebrate/invertebrate Gα subunits and fluoroaluminates, were detected. In one- and two-dimensional immunoblot analyses, an antiserum directed against Giα-type subunits exhibited cross-reactivity at 42 kD, whereas a 43-kD protein cross-reacted with antisera directed against Gqα subunits. Green light below 1 μE m−2 sec−1 suppressed cholera toxin–dependent ADP ribosylation at these apparent molecular masses and modulated a significant proportion of the GTPase activity in a reversible manner. Antisera against Chlamydomonas rhodopsin and the Gα subunits completely impaired light modulation. Both light sensitivity and dark recovery of the GTPase were affected by changes in free Ca2+. Dissociation of the putative Gα subunits from the eyespot membranes was not observed when the membranes were illuminated. Our results emphasize the regulatory potential of Gα subunits in rhodopsin-based signaling of flagellate green algae. INTRODUCTION Phototaxis, a directional movement response of flagellate algae with respect to light direction and intensity, is the basis for orientation toward areas that match best the individual irradiation requirements of the algae. A central constituent of the visual system for this response in green algae is the eyespot apparatus, which is a complex “organelle” involving local specializations of membranes from different subcellular compartments. The functional eyespot apparatus consists of the eyespot plate, that is, one to several layers of carotenoid-rich lipid globules located within the chloroplast and often associated with a specialized thylakoid, and the adjacent specialized areas of the chloroplast envelope and plasma membrane. By combining shading and interference reflector properties, this organelle acts as a directional antenna, thereby enhancing the precision of the phototactic response (Foster and Smyth, 1980; Kreimer and Melkonian, 1990; Kreimer, 1994). Phototaxis is influenced in a complex manner by the concentration of Ca2+ in the medium and occurs over several orders of magnitude of light intensities (Morel-Laurens, 1987; Zacks and Spudich, 1994), pointing to fine-tuned regulation. In contrast to the well-characterized responses and possible signaling elements of the flagella (reviewed in Witman, 1993), relatively little is known about the parts of the transduction chain toward the flagella downstream of the photoreceptor or involved in adaptational processes. The chromophore of the photoreceptor for phototaxis in green algae is all-trans-retinal. Rhodopsin is most likely localized in the plasma membrane patch overlying the eyespot (Foster et al., 1984; Beckmann and Hegemann, 1991; Kreimer et al., 1991b; Deininger et al., 1995). Excitation of the receptor by blue-green light triggers rapid opening of ion channels in the region of the eyespot apparatus, which in turn initiates signal spread toward the flagella (Litvin et al., 1978; Harz and Hegemann, 1991; Beck and Uhl, 1994). The photoreceptor current in the eyespot region is mainly carried by Ca2+ (Nonnengässer et al., 1996) and is graded with the light intensity. The intensity-dependent component of the current is triggered after a 120- to 400-μsec lag period, which has led to the suggestion that rhodopsin and the ion channel form a complex (Sineshchekov et al., 1990). In Chlamydomonas, the cDNA for rhodopsin is currently the only element of the signal transduction chain in the eyespot region that has been characterized (Deininger et al., 1995). In addition, several genes of the general pathway have been identified, with most of their products probably being involved in flagellar responses (Horst and Witman, 1993; Pazour et al., 1995). Based on the rapid occurrence of the photoreceptor current upon intense light flashes and some sequence similarities to voltage/nucleotide–gated ion channels, Deininger et al. (1995) have suggested that in Chlamydomonas, rhodopsin might function as a light-gated ion channel. However, the unusual stimulus–response curves of the photoreceptor peak current and its biphasic decay, as well as rapid adaptational effects of its amplitude to repetitive flashing, suggest the presence of additional distinct cellular control mechanisms of the ion channel(s) involved in photoreception (Sineshchekov et al., 1990; Harz et al., 1992). In addition, a recent kinetic analysis of photocurrents in Chlamydomonas under conditions in which only one or a few photons are absorbed per cell points to the possibility that bleaching of a single rhodopsin molecule might activate more than one Ca2+ channel in the eyespot region. The sigmoidal rising phase of the photoreceptor current under low-light conditions and the fact that the kinetics quicken with increasing photon exposure are consistent with a collision-coupling mechanism by which the rhodopsin activates several calcium channels (Beck, 1996; R. Uhl, personal communication). Whether such a coupling would occur via a direct collision between rhodopsin and the channel(s) or via additional elements, such as heterotrimeric guanine nucleotide binding proteins (G proteins), cannot be determined from these data. In plants, knowledge of G proteins and their physiological functions is still very limited, although Gα- and Gβ-like proteins have been identified in higher plants as well as in Chlamydomonas (Schloss, 1990; Terryn et al., 1993; Ma, 1994; von Kampen et al., 1994; Gotor et al., 1996; Millner and Causier, 1996; Weiss et al., 1997). Physiological functions in which they most likely play a central role include control of Ca2+ channel activity, blue and red light responses of higher plants, as well as activation of phospholipases C and D in Chlamydomonas spp or the adenylyl cyclase in Euglena (Warpeha et al., 1991; Yueh and Crain, 1993; Millar et al., 1994; Munnik et al., 1995; Schumaker and Gizinski, 1996; Torres-Márquez et al., 1996; Wu et al., 1996). Several lines of evidence also point to the presence of G proteins in eyespot preparations of green algae, although their function as putative regulatory elements in phototaxis remains controversial (Korolkov et al., 1990; Hegemann and Harz, 1993; Kreimer, 1994; Deininger et al., 1995). Chlamydomonas eyespot preparations have the potential to activate bovine transducin (Dumler et al., 1989), and specific enrichment of at least two putative Gα subunits (43 and 54 kD) has been recently demonstrated for eyespot preparations of the green alga Spermatozopsis (Schlicher et al., 1995). Here, we present further evidence for the presence of heterotrimeric G proteins in eyespot preparations and demonstrate specific, reversible green light modulation of at least one Gα subunit. In addition, evidence for feedback regulation of the activated Gα subunit(s) by free Ca2+ is presented. RESULTS The Eyespot Apparatus of Spermatozopsis As shown in Figures 1Aand 1B, the eyespot apparatus of Spermatozopsis is located in an anterior lobe of a single, large chloroplast. The distance between the eyespot apparatus and the flagella is ~1 to 1.5 μm. It consists of a single row of carotenoid-rich lipid globules closely attached to the eyespot membranes (Preisig and Melkonian, 1984; Figures 1Aand 1B). Due to the exposed location of the eyespot apparatus as well as the natural lack of a rigid cell wall in Spermatozopsis, this alga was used to devise a simple procedure for the mass isolation of structurally well-preserved eyespot apparatuses (Figures 1Cand 1D; Kreimer et al., 1991a). No sealed structures are formed; therefore, different treatments (e.g., inhibitor and antibody incubations) are possible without the use of permeabilizing agents. All experiments described in this study were conducted with isolated eyespot apparatuses. Figure 1. Open in new tabDownload slide The Eyespot Apparatus of Spermatozopsis. (A) Differential interference contrast image of a living, immobilized cell. The arrow indicates the position of the eyespot. (B) Longitudinal section showing the localization of the single-layered eyespot apparatus in a lobe of the chloroplast. bb, basal body; ey, eyespot apparatus; arrow, flagellar root. (C) and (D) Isolated eyespot apparatuses. Bar in (A) = 10 μm; bar in (B) = 500 nm; bars in (C) and (D) = 200 nm. Figure 1. Open in new tabDownload slide The Eyespot Apparatus of Spermatozopsis. (A) Differential interference contrast image of a living, immobilized cell. The arrow indicates the position of the eyespot. (B) Longitudinal section showing the localization of the single-layered eyespot apparatus in a lobe of the chloroplast. bb, basal body; ey, eyespot apparatus; arrow, flagellar root. (C) and (D) Isolated eyespot apparatuses. Bar in (A) = 10 μm; bar in (B) = 500 nm; bars in (C) and (D) = 200 nm. Figure 2. Open in new tabDownload slide High-Affinity GTP Binding in Isolated Eyespot Apparatuses of Spermatozopsis. The displacement of 35S-GTP-γ-S binding by increasing concentrations of various unlabeled nucleotides was measured at a fixed 35S-GTP-γ-S concentration of 10−9 M. Data represent the mean of two independent isolations. Figure 2. Open in new tabDownload slide High-Affinity GTP Binding in Isolated Eyespot Apparatuses of Spermatozopsis. The displacement of 35S-GTP-γ-S binding by increasing concentrations of various unlabeled nucleotides was measured at a fixed 35S-GTP-γ-S concentration of 10−9 M. Data represent the mean of two independent isolations. 35S-GTP-γ-S Binding and GTPase Activity in Isolated Eyespot Apparatuses Specific high-affinity 35S-GTP-γ-S binding was detected in Spermatozopsis eyespot fractions. Figure 2shows that binding was effectively suppressed by similar concentrations of unlabeled GTP and GDP, as is known for many G proteins (e.g., Northup et al., 1982; Grand and Owen, 1991). Half-maximal inhibition was observed at ~10−7 M. Neither ATP nor CTP had a significant effect in this concentration range. In addition, the preparations exhibited high-affinity GTPase activity (Km app of ~4 μM); substrate saturation was observed at ~20 μM GTP. The activity was strictly Mg2+ dependent and was not affected by a 400-fold excess of App(NH)p, a nonhydrolyzable ATP analog. However, the same excess of the nonhydrolyzable or poorly hydrolyzable GTP analogs Gpp(NH)p and GTP-γ-S suppressed 50 to 80% of the GTPase activity. GTPase activity was not stable. Storage at either 4 or −20°C resulted in a rapid decrease of the specific GTPase activity. To measure preferentially specific high-affinity GTPases, standard assays were conducted at low substrate concentrations (0.5 μM) within 24 hr after isolation of the eyespot apparatuses. The average measured GTPase activity under these conditions varied between 4 and 9 pmol of GDP mg −1 of protein sec−1 (n = 54 independent eyespot preparations) and was thus slightly higher than was the activity reported for Chlamydomonas eyespot fractions (Korolkov et al., 1990). These values certainly reflect a mixture of different GTP hydrolyzing activities, because more than one putative Gα subunit as well as small GTP binding proteins are present in the preparations (Kreimer, 1994; Schlicher et al., 1995). In a first attempt to differentiate between these two groups of G proteins, we analyzed the effect of fluoroaluminates (AlF) on their activity. AlF are reported to interact with heterotrimeric G proteins but not with small monomeric G proteins or ADP ribosylation factors. The complexes bind to the GDP-bound form of Gα subunits, leading to permanent activation of the G proteins and thus to suppression of their GTP hydrolyzing activity (Kahn, 1991; Pennington, 1994). In the presence of 20 μM AlF, an average reduction in GDP formation by 43.5 ± 10.8% was observed in the eyespot fraction. Inhibition was rapid, and preincubation with AlF in the absence of GTP for 5 min resulted in no significant additional increase in average inhibition. To confirm further that this proportion of the total GTPase activity might be due to heterotrimeric G proteins, we used peptide antibodies raised against vertebrate/invertebrate Gα subunits. Table 1summarizes that when compared with AlF, a somewhat lower proportion of total GTPase activity was suppressed by the different Gα antisera tested. All of these antisera exhibited cross-reactivity with eyespot fraction proteins when subjected to immunoblot analyses (Figure 3). Average inhibition varied between 22 and 27% and depended on the concentration of the antiserum (Table 1). A significantly lower effect on GTP hydrolysis was observed in corresponding preimmune controls. As an additional control, different antisera directed against proteins not specifically enriched in the eyespot apparatuses were used. Again, no significant inhibition was caused by these antisera (average inhibition was 2 to 5%). Inhibition efficiency was identical between the antiserum directed against the C terminus of Giα subunits (MB-1) and the peptide antisera directed against the C or N terminus of Table 1. Effects of Different Peptide Antisera Directed against Mammalian/Invertebrate Gα Subunits on Total GTPase Activity in Isolated Eyespot Apparatuses of Spermatozopsis Antiseruma . GTPase Inhibition (%) . Giα', C terminus (1:100) 25.8 ± 6.4 Giα', C terminus (1:1000) 12.7b Giα', C terminus preimmune (1:100) 4.5 ± 7.3 Giα', C terminus no preincubation (1:100) 13.5 ± 6.8 Gqα', N terminus (1:100) 26.5 ± 7.3 Gqα', C terminus (1:100) 21.6 ± 6.5 Antiseruma . GTPase Inhibition (%) . Giα', C terminus (1:100) 25.8 ± 6.4 Giα', C terminus (1:1000) 12.7b Giα', C terminus preimmune (1:100) 4.5 ± 7.3 Giα', C terminus no preincubation (1:100) 13.5 ± 6.8 Gqα', N terminus (1:100) 26.5 ± 7.3 Gqα', C terminus (1:100) 21.6 ± 6.5 a Preincubation was done, if not otherwise stated, at room temperature for 20 min. Data are from four or five independent eyespot isolations ±SD.The antiserum MB-1 was used as a probe for Giα', C terminus. b Mean of two determinations. Open in new tab Table 1. Effects of Different Peptide Antisera Directed against Mammalian/Invertebrate Gα Subunits on Total GTPase Activity in Isolated Eyespot Apparatuses of Spermatozopsis Antiseruma . GTPase Inhibition (%) . Giα', C terminus (1:100) 25.8 ± 6.4 Giα', C terminus (1:1000) 12.7b Giα', C terminus preimmune (1:100) 4.5 ± 7.3 Giα', C terminus no preincubation (1:100) 13.5 ± 6.8 Gqα', N terminus (1:100) 26.5 ± 7.3 Gqα', C terminus (1:100) 21.6 ± 6.5 Antiseruma . GTPase Inhibition (%) . Giα', C terminus (1:100) 25.8 ± 6.4 Giα', C terminus (1:1000) 12.7b Giα', C terminus preimmune (1:100) 4.5 ± 7.3 Giα', C terminus no preincubation (1:100) 13.5 ± 6.8 Gqα', N terminus (1:100) 26.5 ± 7.3 Gqα', C terminus (1:100) 21.6 ± 6.5 a Preincubation was done, if not otherwise stated, at room temperature for 20 min. Data are from four or five independent eyespot isolations ±SD.The antiserum MB-1 was used as a probe for Giα', C terminus. b Mean of two determinations. Open in new tab Figure 3. Open in new tabDownload slide Immunoblot Analysis of the Eyespot Fraction after SDS-PAGE and Two-Dimensional Gel Electrophoresis with Antisera Directed against Vertebrate/Invertebrate Gα Subunits. (A) Lane 1 contains protein stained with Coomassie Brilliant Blue R 250; lane 2, antiserum MB-1, which is directed against the C-terminal peptide KENLKDCGLF common in Giα; lane 3, antiserum directed against a C-terminal peptide common to squid and mouse Gqα (KDTILQLNLKEYNLV); lane 4, antiserum directed against the N-terminal peptide CLSEEAKEQKRINQE of Drosophila Gqα; and lane 5, preimmune to MB-1. The same amount of protein (25 μg) was applied to the gels. The antisera were diluted 1:3000. Numbers at left indicate apparent molecular masses in kilodaltons. (B) Immunoblot analysis after two-dimensional gel electrophoretic analysis. At top is an immunoblot probed with an antiserum directed against a C-terminal peptide common to squid and mouse Gqα (1:3000). At bottom is an immunoblot probed with the Giα antiserum MB-1 (1:6000). The arrows indicate the 43- and 42-kD proteins, respectively. The molecular mass markers are indicated at left in kilodaltons. Figure 3. Open in new tabDownload slide Immunoblot Analysis of the Eyespot Fraction after SDS-PAGE and Two-Dimensional Gel Electrophoresis with Antisera Directed against Vertebrate/Invertebrate Gα Subunits. (A) Lane 1 contains protein stained with Coomassie Brilliant Blue R 250; lane 2, antiserum MB-1, which is directed against the C-terminal peptide KENLKDCGLF common in Giα; lane 3, antiserum directed against a C-terminal peptide common to squid and mouse Gqα (KDTILQLNLKEYNLV); lane 4, antiserum directed against the N-terminal peptide CLSEEAKEQKRINQE of Drosophila Gqα; and lane 5, preimmune to MB-1. The same amount of protein (25 μg) was applied to the gels. The antisera were diluted 1:3000. Numbers at left indicate apparent molecular masses in kilodaltons. (B) Immunoblot analysis after two-dimensional gel electrophoretic analysis. At top is an immunoblot probed with an antiserum directed against a C-terminal peptide common to squid and mouse Gqα (1:3000). At bottom is an immunoblot probed with the Giα antiserum MB-1 (1:6000). The arrows indicate the 43- and 42-kD proteins, respectively. The molecular mass markers are indicated at left in kilodaltons. invertebrate Gqα (Table 1). In contrast to AlF, the effect of the antisera strongly depended on the preincubation time. Compared with assays without preincubation, assays with preincubation (20 min) of the eyespot apparatuses with the antiserum MB-1 showed significantly higher inhibition. However, the effects of the Gα antisera were still somewhat rapid. Within 90 sec, an average decrease in GDP formation of 14% was observed for MB-1 and other antisera. Thus, even in the isolated eyespot apparatuses (Figures 1Cand 1D), possible sterical hindrance due to a more or less close attachment of the eyespot membranes did not appear to be a major problem. However, this may well be a reason for the on average lower inhibition induced by the antisera compared with the AlF complexes. Immunodetection of G Proteins All antisera affecting the GTPase activity also showed cross-reactivity with proteins of the Spermatozopsis eyespot fraction in the known molecular mass range of Gα subunits (36 to 56 kD; Figure 3). Additional cross-reactive proteins of higher (>56 kD) and lower (<32 kD) apparent molecular masses were detected, especially by antiserum MB-1. However, their relative abundance was somewhat variable (e.g., see Figure 3Band Schlicher et al. [1995]). In the gel system used in our study, the MB-1 cross-reactive protein, which has recently been shown to be specifically enriched in the eyespot fraction (Schlicher et al., 1995), migrated at 42 kD (Figure 3A, lane 2). In addition, a protein of slightly higher apparent molecular mass (43 kD) was labeled by two Gqα peptide antisera (Figure 3A, lanes 3 and 4). The corresponding protein stains revealed that the cross-reactive proteins migrated slightly below two main Coomassie blue–stained bands, thus representing only minor constituents of the eyespot apparatus (Figure 3A, lane 1). Two-dimensional gel electrophoresis demonstrated that the cross-reactive proteins at 42 and 43 kD focused at clearly different pH values (Figure 3B). The Gqα peptide antiserum recognized a 43-kD protein with a pI of ~5.9 to 6.1. The MB-1 cross-reactive protein at 42 kD exhibited a clearly more acidic isoelectric point (~4.9 to 5.1). No specific labeling at 42 and 43 kD was observed with a commercially available peptide antiserum raised against the C terminus of stimulatory Gα subunits (amino acids 385 to 394; data not shown). ADP Ribosylation by Cholera Toxin Plant Gα subunits contain ADP ribosylation sites for bacterial toxins which include cholera toxin (CTX; Ma, 1994). To confirm further that the cross-reactive proteins in our eyespot fraction represent Gα subunits, CTX-dependent ADP ribosylation was conducted. The protein(s) at 42/43 kD was a major CTX substrate in the fraction and was rapidly labeled (Figure 4). In the absence of CTX, only a low molecular mass protein was labeled. Excess arginine as well as increasing amounts of unlabeled NAD inhibited the incorporation of radioactivity in all bands (data not shown), further indicating the specificity of labeling by CTX. Storage at either 4 or −20°C resulted in a complete loss of CTX-dependent ADP ribosylation at 42/43 kD. An unequivocal assignment of the 32P-labeled band to either the 43- or 42-kD protein was not possible. Effect of Light on GTPase Activity, ADP Ribosylation, and Membrane Association of the Gα Subunits To assign a role to the putative heterotrimeric G proteins in the chain of events during photoresponses, we investigated light regulation of the GTPase(s). We first analyzed the effect of phototactic active green and photosynthetic active red light at low fluence rates on total GTPase activity (Figure 5A). In comparison to the dark control, broad-band green light (490 to 570 nm) inhibited GTPase activity in a fluence rate–dependent manner. A significant reduction in GDP formation was already apparent at 0.01 μE m−2 sec−1. At this fluence rate, algae such as Chlamydomonas start to show Figure 4. Open in new tabDownload slide CTX-Dependent ADP Ribosylation of Proteins in the Eyespot Fraction. 32P-ADP–ribosylated eyespot proteins (30 μg) were separated by SDS-PAGE (12.5%) and autoradiographed. t5 and t30, complete assays with 5- and 30-min incubation, respectively; −CTX, control without CTX. Arrows indicate the 42/43-kD protein(s). Numbers at left indicate apparent molecular masses in kilodaltons. Figure 4. Open in new tabDownload slide CTX-Dependent ADP Ribosylation of Proteins in the Eyespot Fraction. 32P-ADP–ribosylated eyespot proteins (30 μg) were separated by SDS-PAGE (12.5%) and autoradiographed. t5 and t30, complete assays with 5- and 30-min incubation, respectively; −CTX, control without CTX. Arrows indicate the 42/43-kD protein(s). Numbers at left indicate apparent molecular masses in kilodaltons. strong phototactic orientation (Pazour et al., 1995). Inhibition increased linearly up to 1 μE m−2 sec−1. A further increase in fluence rate resulted in additional inhibition that was minor (data not shown). Red light had only a negligible inhibitory effect, with no clear fluence rate dependence (Figure 5A). Closer analysis of spectral dependence, using monochromatic filters, revealed maximal inhibition at 502 nm (Figure 5B). However, the samples were extremely rich in carotenoids (Kreimer et al., 1991a), which severely affects the real spectral dependence toward the blue region. Green light modulation in the in vitro system of isolated eyespot apparatuses was almost completely reversible. Preilluminated samples kept in the dark for 3 to 5 min exhibited almost complete recovery of GTPase activity. Light modulation of GTPase activity was rapid. On average, 80% of the effect was manifested within the first 30 sec of illumination with green light (Figure 5C). In contrast, the red light–mediated effect was slow and was manifested only at longer illumination times. In addition, the red light–induced inhibition was insensitive to storage, whereas sensitivity toward blue-green light rapidly decreased when stored both at 4 or −20°C concomitant with the general loss of specific Gpp(NH)p-sensitive GTPase activity. Thus, the red light effect can most likely be ascribed to a second light-absorbing system, for example, contaminating chlorophyll, or to an unspecific effect. The percentage of light-sensitive GTPase activity (22 to 47% of total activity) was in the range of that affected by AlF and Gα-specific antisera, indicating that at least one of the cross-reactive proteins might be the target of light modulation. As shown in Table 2, further support for this assumption arises from the inhibitory effects of the Gα antisera on the green light modulation. Whereas the preimmune control exhibited a normal green light modulation, no additional light-induced inhibition occurred in the presence of the different Gα antisera. Because this effect could be due either to a specific disturbance of the signaling system or to an unspecific general disturbance by antigen/antibody complex formation, we also analyzed the effect of light on ADP ribosylation (Figure 6). At low fluence rates (0.01 μE m−2 sec−1), green light clearly reduced the labeling intensity of the band at 42/43 kD, whereas red light had no effect (Figure 6A). Quantitative analysis revealed an average reduction in the labeling intensity at these molecular masses of 68% by green light at 1 μE m−2 sec−1 (Figure 6B). Red light at the same fluence rate reduced the labeling on average by 30%. The slightly higher effect of red light on ADP ribosylation than on GTPase modulation is probably due to the prolonged illumination time (30 min versus 90 sec). Labeling of other major CTX substrates, as exemplified for the 24-kD protein, was not affected by green light (Figure 6B). Immunoblot analysis revealed that when illuminated (30 μE m−2 sec−1, 10 min at 4°C) with white or green light in the presence of GTP and AlF, neither the 42- nor the 43-kD protein was released from the eyespot membranes (data not shown). Figure 5. Open in new tabDownload slide Light Modulation of GTPase Activity in Isolated Eyespot Apparatuses. (A) Effect of increasing fluence rates of photosynthetic active red light (>560 nm) and phototactic active green light (490 to 570 nm) on total GTPase activity. The eyespot fractions were illuminated as indicated during the assay. n = 3 independent eyespot preparations ±sd. (B) Spectral analysis of the light modulation with monochromatic filters. The fluence rate was adjusted to 1 μE m−2 sec−1. Note that due to the high carotenoid content of the sample, spectral dependence was severely affected in the blue region. n = 11 to 14 independent eyespot preparations ±sd. (C) Time course of light-induced inhibition of GTPase activity. The following filters were used: green at 502 nm and red at 663 nm. The fluence rate was adjusted to 1 μE m−2 sec−1. Data represent the mean of six independent isolations ±sd. Figure 5. Open in new tabDownload slide Light Modulation of GTPase Activity in Isolated Eyespot Apparatuses. (A) Effect of increasing fluence rates of photosynthetic active red light (>560 nm) and phototactic active green light (490 to 570 nm) on total GTPase activity. The eyespot fractions were illuminated as indicated during the assay. n = 3 independent eyespot preparations ±sd. (B) Spectral analysis of the light modulation with monochromatic filters. The fluence rate was adjusted to 1 μE m−2 sec−1. Note that due to the high carotenoid content of the sample, spectral dependence was severely affected in the blue region. n = 11 to 14 independent eyespot preparations ±sd. (C) Time course of light-induced inhibition of GTPase activity. The following filters were used: green at 502 nm and red at 663 nm. The fluence rate was adjusted to 1 μE m−2 sec−1. Data represent the mean of six independent isolations ±sd. Table 2. Effects of Different Antisera Directed against Mammalian/Invertebrate Gα Subunits on Green Light Modulation of Total GTPase Activity in Isolated Eyespot Apparatuses of Spermatozopsis . Inhibition of Total GTPase (%) . Antiserum . Dark . Green Lighta . None 0 25.3 ± 4.0 Giα', C terminus preimmune (1:100) 4.5 ± 7.3 26.8 ± 1.8 Giα', C terminus (1:100) 25.8 ± 6.4 28.0 ± 6.0 Gqα' N terminus (1:100) 26.5 ± 7.3 26.7 ± 6.1 Gqα' C terminus (1:100) 21.6 ± 6.5 25.5 ± 4.9 . Inhibition of Total GTPase (%) . Antiserum . Dark . Green Lighta . None 0 25.3 ± 4.0 Giα', C terminus preimmune (1:100) 4.5 ± 7.3 26.8 ± 1.8 Giα', C terminus (1:100) 25.8 ± 6.4 28.0 ± 6.0 Gqα' N terminus (1:100) 26.5 ± 7.3 26.7 ± 6.1 Gqα' C terminus (1:100) 21.6 ± 6.5 25.5 ± 4.9 a Illumination conditions included broad-band filters of 490 to 570 nm at 1 μE m−2 sec−1 for 90 sec. Data are from four or five independent isolations ±sd. All other conditions were as described in Table 1. Open in new tab Table 2. Effects of Different Antisera Directed against Mammalian/Invertebrate Gα Subunits on Green Light Modulation of Total GTPase Activity in Isolated Eyespot Apparatuses of Spermatozopsis . Inhibition of Total GTPase (%) . Antiserum . Dark . Green Lighta . None 0 25.3 ± 4.0 Giα', C terminus preimmune (1:100) 4.5 ± 7.3 26.8 ± 1.8 Giα', C terminus (1:100) 25.8 ± 6.4 28.0 ± 6.0 Gqα' N terminus (1:100) 26.5 ± 7.3 26.7 ± 6.1 Gqα' C terminus (1:100) 21.6 ± 6.5 25.5 ± 4.9 . Inhibition of Total GTPase (%) . Antiserum . Dark . Green Lighta . None 0 25.3 ± 4.0 Giα', C terminus preimmune (1:100) 4.5 ± 7.3 26.8 ± 1.8 Giα', C terminus (1:100) 25.8 ± 6.4 28.0 ± 6.0 Gqα' N terminus (1:100) 26.5 ± 7.3 26.7 ± 6.1 Gqα' C terminus (1:100) 21.6 ± 6.5 25.5 ± 4.9 a Illumination conditions included broad-band filters of 490 to 570 nm at 1 μE m−2 sec−1 for 90 sec. Data are from four or five independent isolations ±sd. All other conditions were as described in Table 1. Open in new tab Involvement of a Putative Spermatozopsis Rhodopsin in Light Modulation of the GTPase The observed maximal sensitivity of the light modulation of the GTPase close to 500 nm is in good agreement with the spectral data available on green algal rhodopsins (Beckmann and Hegemann, 1991; Kreimer et al., 1991b). To gain additional evidence for the close coupling of at least one of the GTPases to this photoreceptor, we have used an antiserum directed against Chlamydomonas rhodopsin. Chlamydomonas rhodopsin migrates in SDS-PAGE analysis at ~30 kD. It is believed to be the only photoreceptor for both photoshock and phototactic responses (Kröger and Hegemann, 1994; Deininger et al., 1995). In eyespot preparations of Spermatozopsis, the antiserum against Chlamydomonas rhodopsin strongly cross-reacted with a 32-kD protein (Figure 7A, lane 2). Immunoblot analysis of different subcellular fractions of Spermatozopsis revealed specific enrichment of this protein in the eyespot fraction (Figure 7B). In the same molecular mass range, 3H-retinal labeling was observed (Figure 7A, lane 4), further indicating that the antiserum might also recognize the putative rhodopsin in Spermatozopsis. Thus, we analyzed the effect of this antiserum on the GTPase activity and its green light modulation (Figure 7C). Preincubation of the eyespot fraction with this antiserum significantly interfered with GTPase activity. GDP hydrolysis was already reduced in the dark to the same level as when illuminated. No further increase in inhibition was observed when the eyespot fraction was treated with green light, whereas the corresponding preimmune serum still facilitated green light modulation at the same magnitude as did the control (Figure 7C). Ca2+ Effects on Light Modulation of the GTPase Ca2+ is intricately and deeply involved at different steps in photoorientation (reviewed in Witman, 1993). To investigate the possible involvement of Ca2+ in our system, we analyzed both light modulation and dark recovery kinetics of GTPase activity (Figure 8). First, the effect of varying free Ca2+ concentrations on light modulation was analyzed (Figure 8A). Suppression of GTPase activity by green light increased with the concentration of free Ca2+ in the assay medium. Comparison of the mean inhibition observed at 10−5 M Ca2+ with that at 10−7 M revealed a P value <0.0001, whereas the P value for red light modulation was >0.05. Upon lowering of free Ca2+ to very low levels (10−9 M), no light modulation was detectable by using our assay system. Green light modulation might have been superimposed by a general increase in GTP hydrolysis, which was observed in both illuminated and dark-kept samples at this reduced Ca 2+ Figure 6. Open in new tabDownload slide Green Light–Reduced CTX-Dependent ADP Ribosylation of the 42/43-kD Protein(s). (A) ADP ribosylation of eyespot proteins was conducted simultaneously in darkness, red light (663 nm), or green light (490 to 570 nm) for 30 min. The fluence rates were adjusted to 0.01 μE m−2 sec−1. Only the relevant regions of the gel are shown. The apparent molecular mass is indicated at left. (B) Quantitative analysis of the effects of red (663 nm) and green (502 nm) illumination on the relative (rel.) ADP ribosylation intensities of the 42/43- and 24-kD proteins. The fluence rates were 1 μE m−2 sec−1. The 24-kD protein served as internal control to rule out possible unspecific effects of illumination on the toxin. n = 6 (red light) and 14 (green light) independent eyespot isolations ±sd. Figure 6. Open in new tabDownload slide Green Light–Reduced CTX-Dependent ADP Ribosylation of the 42/43-kD Protein(s). (A) ADP ribosylation of eyespot proteins was conducted simultaneously in darkness, red light (663 nm), or green light (490 to 570 nm) for 30 min. The fluence rates were adjusted to 0.01 μE m−2 sec−1. Only the relevant regions of the gel are shown. The apparent molecular mass is indicated at left. (B) Quantitative analysis of the effects of red (663 nm) and green (502 nm) illumination on the relative (rel.) ADP ribosylation intensities of the 42/43- and 24-kD proteins. The fluence rates were 1 μE m−2 sec−1. The 24-kD protein served as internal control to rule out possible unspecific effects of illumination on the toxin. n = 6 (red light) and 14 (green light) independent eyespot isolations ±sd. concentration. A similar effect of Ca2+ on basal dark activity was not evident at higher free Ca2+ concentrations (Figure 8A). Therefore, to determine whether this effect was due to a loss in light modulation at low free Ca2+ or to limitations of the assay is not possible. However, an increase in GTP hydrolysis is indicative of enhanced deactivation of GTPases (Pennington, 1994). Because the threshold for Ca2+-induced basal body reorientation in Spermatozopsis points to a free cytosolic Ca 2+ concentration in nonstimulated cells below 10−7 M (McFadden et al., 1987), the effect of Ca2+ concentrations clearly above and below this critical concentration on the dark recovery of the GTPase was analyzed. Eyespot samples were illuminated at 10−5 M free Ca2+ for 90 sec and then adjusted to either 10−9 M or kept at 10−5 M Ca2+ for various time periods. Samples were kept in complete darkness before the GTPase assay (Figure 8B). At elevated concentrations of free Ca2+ in the dark, the recovery of activity was slow and was completed after ~210 sec. In contrast, when Ca2+-sequestering activities were mimicked by lowering the concentration of this ion to 10−9 M, the GTPase activity was completely restored without additional dark incubation, that is, within the assay time of 90 sec (Figure 8B). DISCUSSION Dissection of the phototactic signaling pathway(s) in the eyespot region of flagellate green algae at the molecular level has recently started with the cloning of the cDNA for rhodopsin and the application of insertional mutagenesis (Deininger et al., 1995; Pazour et al., 1995). In green algae, rhodopsin-mediated signaling leads not only to phototaxis and photoshock (reviewed in Witman, 1993; Kreimer, 1994; Hegemann, 1997) but also triggers retinal synthesis (Foster et al., 1988). Known rapid adaptational responses of green algae over an extreme intensity range (Sineshchekov et al., 1990; Zacks and Spudich, 1994) must also be finally coupled to excitation of the rhodopsin. Thus, complex and divergent signaling pathways can be anticipated to be initiated by its activation. The presence of more than one putative Gα subunit in eyespot preparations of Chlamydomonas (Korolkov et al., 1990; Hegemann and Harz, 1993) and Spermatozopsis (Schlicher et al., 1995; this study) is therefore not surprising. The apparent molecular masses of the putative Gα subunits of Spermatozopsis are well within the typical molecular mass range (35 to 56 kD) reported for Gα subunits of vertebrates/invertebrates and higher plants (Ma, 1994; Pennington, 1994; Gotor et al., 1996). In addition, GTPase activity exhibits general biochemical characteristics of heterotrimeric GTPases. However, basal GTPase activities in green algal eyespot preparations are higher than in most membrane preparations of the eyes of vertebrates and invertebrates (Korolkov et al., 1990; this study). The reasons for this are currently not known. Figure 7. Open in new tabDownload slide Involvement of a Putative Spermatozopsis Rhodopsin in Light Modulation of the GTPase(s). (A) Immunoblot (lanes 1 to 3) analysis with an antiserum directed against Chlamydomonas rhodopsin and 3H-retinal binding analysis (lane 4) revealed the presence of a putative rhodopsin in the eyespot fraction of Spermatozopsis. Lane 1 contains eyespot proteins stained with Coomassie blue; lane 2, eyespot apparatuses probed with a polyclonal antiserum directed against the rhodopsin of Chlamydomonas; and lane 3, preimmune control. Twenty-five micrograms of protein was loaded on the gel, and the antiserum dilutions were 1:5000. Lane 4 shows autoradiography of the eyespot fraction incubated with 3H-retinal and separated by gradient SDS-PAGE (11 to 17%). One hundred micrograms of protein was loaded on the gel. Numbers at left indicate apparent molecular masses in kilodaltons. (B) Immunoblot analysis of different fractions of Spermatozopsis revealed specific enrichment of the putative photoreceptor in the eyespot apparatuses. Lane 1 contains the crude extract; lane 2, the eyespot fraction; lane 3, soluble proteins; lane 4, a fraction enriched in plasma membrane vesicles. The dilution of the serum was 1:5000. Approximately 25 μg of protein was loaded on the gel, and developing times were identical for all fractions. (C) Effect of the Chlamydomonas rhodopsin antiserum on light modulation of the GTPase is shown. The eyespot samples were incubated for 20 min with the antiserum (1:100) or preimmune (1:100) serum before the assays. Assays were either done in darkness or illuminated with green light. Data are the mean of four independent isolations ±sd. The fluence rate of green light (502 nm) was adjusted to 1 μE m−2 sec−1. Figure 7. Open in new tabDownload slide Involvement of a Putative Spermatozopsis Rhodopsin in Light Modulation of the GTPase(s). (A) Immunoblot (lanes 1 to 3) analysis with an antiserum directed against Chlamydomonas rhodopsin and 3H-retinal binding analysis (lane 4) revealed the presence of a putative rhodopsin in the eyespot fraction of Spermatozopsis. Lane 1 contains eyespot proteins stained with Coomassie blue; lane 2, eyespot apparatuses probed with a polyclonal antiserum directed against the rhodopsin of Chlamydomonas; and lane 3, preimmune control. Twenty-five micrograms of protein was loaded on the gel, and the antiserum dilutions were 1:5000. Lane 4 shows autoradiography of the eyespot fraction incubated with 3H-retinal and separated by gradient SDS-PAGE (11 to 17%). One hundred micrograms of protein was loaded on the gel. Numbers at left indicate apparent molecular masses in kilodaltons. (B) Immunoblot analysis of different fractions of Spermatozopsis revealed specific enrichment of the putative photoreceptor in the eyespot apparatuses. Lane 1 contains the crude extract; lane 2, the eyespot fraction; lane 3, soluble proteins; lane 4, a fraction enriched in plasma membrane vesicles. The dilution of the serum was 1:5000. Approximately 25 μg of protein was loaded on the gel, and developing times were identical for all fractions. (C) Effect of the Chlamydomonas rhodopsin antiserum on light modulation of the GTPase is shown. The eyespot samples were incubated for 20 min with the antiserum (1:100) or preimmune (1:100) serum before the assays. Assays were either done in darkness or illuminated with green light. Data are the mean of four independent isolations ±sd. The fluence rate of green light (502 nm) was adjusted to 1 μE m−2 sec−1. Although a role of G proteins in phototactic signaling of green algae has already been suggested by different authors (reviewed in Kreimer, 1994), light regulation of GTPases as a basic prerequisite was not clearly demonstrated until now. The results presented here strongly indicate close coupling of at least one of the putative G proteins to the photoreceptor of phototaxis in Spermatozopsis. Key findings leading to this conclusion are as follows. Fast, linear, and reversible green light modulation of a significant proportion of the total GTPase activity occurred at light intensities (0.01 to 1 μE m−2 sec−1) sufficient to induce phototactic orientation but not the photoshock response (Harz et al., 1992; Pazour et al., 1995). In addition, the overall spectral dependence (maximum at 502 nm) of light modulation is similar to spectral data of green algal rhodopsins and rhodopsin-mediated responses (Litvin et al., 1978; Foster et al., 1984; Beckmann and Hegemann, 1991; Kreimer et al., 1991b). Light-sensitive GTPase activity was completely abolished by antisera directed against Gq and Gi subunits as well as by an antiserum directed against the rhodopsin of Chlamydomonas, whereas corresponding control sera had no significant effect. CTX-dependent ADP-ribosylation of a 42/43-kD band was specifically reduced by green light in the same fluence rate range as the GTPase activity. A general close spatial relationship between the rhodopsin and the two putative G proteins is further sustained by the equal inhibitory effects of green light and the different antisera directed against either the putative photoreceptor or the Gα subunits on the GTPase. Unspecific effects of the antisera by, for example, hindrance of lateral diffusion of the signaling elements in the plasma membrane patch by antigen–antibody complex formation appear unlikely, because antisera raised against proteins present but not specifically enriched in the eyespot fraction and corresponding preimmune controls had no effect. Apparently, irrespective of the precise mode of action of the antisera, complex formation between any of the putative Gα subunits or the photoreceptor and the antisera specifically disturb the whole signaling pathway. A close spatial relationship of the different signaling components is sustained by the high intramembrane Figure 8. Open in new tabDownload slide Light Modulation and Dark Recovery Can Be Influenced by Free Ca2+. (A) Effects of free Ca2+ on total GTPase activity and light modulation. The indicated free Ca2+ concentrations in the assays were adjusted by EGTA/Ca2+ buffers. Fluence rates of red (663 nm) and green (502 nm) light were 1 μE m−2 sec−1. Data represent the mean ±sd of 11 (502 nm) and three (663 nm) independent preparations. (B) Mimicked Ca2+-sequestering activities greatly enhanced the dark recovery of the light-sensitive proportion of GTPase activity. Samples were illuminated (1 μE m−2 sec−1 of green light) at 10−5 M free Ca2+ for 90 sec and then adjusted to either 10−9 M Ca2+ or kept at 10−5 M Ca2+ for the indicated time periods in complete darkness before the GTPase assay. Data are the mean ±sd of five independent eyespot preparations. Figure 8. Open in new tabDownload slide Light Modulation and Dark Recovery Can Be Influenced by Free Ca2+. (A) Effects of free Ca2+ on total GTPase activity and light modulation. The indicated free Ca2+ concentrations in the assays were adjusted by EGTA/Ca2+ buffers. Fluence rates of red (663 nm) and green (502 nm) light were 1 μE m−2 sec−1. Data represent the mean ±sd of 11 (502 nm) and three (663 nm) independent preparations. (B) Mimicked Ca2+-sequestering activities greatly enhanced the dark recovery of the light-sensitive proportion of GTPase activity. Samples were illuminated (1 μE m−2 sec−1 of green light) at 10−5 M free Ca2+ for 90 sec and then adjusted to either 10−9 M Ca2+ or kept at 10−5 M Ca2+ for the indicated time periods in complete darkness before the GTPase assay. Data are the mean ±sd of five independent eyespot preparations. particle density observed in the eyespot membranes and the rapid occurrence of photoreceptor currents (reviewed in Melkonian and Robenek, 1984; Hegemann, 1997). Our findings are also supported by recent detailed kinetic analyses of the rising phase of the photoreceptor current in Chlamydomonas under single photon absorption conditions. These analyses are indicative of a collision coupling mechanism mediated either by direct collision between the rhodopsin and the channels or by, for example, G proteins (R. Uhl, personal communication). It is not yet known why illumination leads to reduced GTPase activity. Possible explanations would be a loss of the target(s) of the putative GTPases during isolation of eyespot apparatuses or prolonged binding to the target(s) upon illumination. Both would affect the catalytic rate of GTP hydrolysis and could lead to decreased overall GTP hydrolysis. Other, more complex explanations, including involvement of members of the RGS protein superfamily (for regulator of G protein signaling, which accelerates GTPase activities; Koelle, 1997) could be proposed, but currently the information about the eyespot signaling system is not sufficient to explain this observation. Interestingly, a reduced GTP hydrolysis rate in the light has also been reported for squid photoreceptor membranes at Ca2+ concentrations above the physiological resting levels (Fyles et al., 1991). The currently known opsin sequences of green algae (C. reinhardtii and Volvox carteri) do not allow any clear conclusion about whether they belong to the group of G protein coupled receptors. They are characterized by principal differences to all other known opsins. Considerable homology has been observed with opsins of invertebrates, especially Drosophila and the blowfly (Deininger et al., 1995). The DRY/ERY consensus motif, which is involved in G protein binding and is present in all rhodopsins, is missing. However, in general, the lack of this motif does not exclude G protein activation, because the two other subfamilies of G protein–coupled receptors do not possess the DRY/ERY motif (Birnbaumer and Birnbaumer, 1995). On the other hand, a notable motif homology of the algal opsin (HE⋯VEPSKKVSLKS179–191, where underlining indicates identical amino acids and dots indicate missing amino acids) to a sequence found in the third cytosolic loop of invertebrate rhodopsins exists. In vertebrates, this loop is involved in coupling rhodopsin to signaling proteins, and a similar function is also likely to occur in invertebrates (reviewed in Birnbaumer and Birnbaumer, 1995; Gärtner and Towner, 1995). Thus, the currently known green algal opsin sequences neither exclude nor support G protein coupling of this retinal-based receptor type. Similarities to signaling in eyes of invertebrates were also observed in some properties of the light modulation of the GTPase(s). In membrane preparations of blowfly eyes, CTX-dependent ADP ribosylation of a putative Gα subunit is suppressed by illumination (Bentrop and Paulsen, 1986), and as in Spermatozopsis, illumination does not induce dissociation of the putative Gα subunit from the membranes. Also, in Arabidopsis and cauliflower, the G protein GPα1 is tightly membrane bound (Weiss et al., 1997). A mainly membrane-delimited signaling pathway in the eyespot membranes would also be more compatible with the observed signaling speed indicated by the current analysis. What might be the functions of the putative G proteins? If the 42-kD protein is indeed similar to the Gi subgroup, which would be in good agreement with the observation that currently known higher plant Gα subunits share considerable homologies to mammalian Giα and Gt (Ma, 1994), it is tempting to speculate about roles in regulation of voltage-gated ion channels. Control of the open/closed probabilities of ion channels by G proteins is a well-known phenomenon in Figure 9. Open in new tabDownload slide Model of Possible Functions for Heterotrimeric G Proteins in Photoresponses of Flagellate Green Algae. The model integrates known elements from Chlamydomonas, Haematococcus, and Spermatozopsis. Photoshock is most likely mediated by a pure electrical signal spread toward the flagella, although involvement of G proteins in signaling from the activated rhodopsin to the PR channel under high and low light conditions cannot be completely ruled out (path 1; see Discussion). Possible functions of G proteins downstream of the PR channel are thus likely to be restricted to the phototactic transduction pathway toward the flagella (path 2). Involvement in light adaptation by control of the probability of opening of ion channels in the vicinity of the eyespot apparatus or the PR channel itself (path 3) would be a third intriguing possibility. By stabilizing the lifetime of the activated Gα subunit(s) at increased cytosolic concentrations or enhancing the deactivation at low free cytosolic concentrations, light-induced concentration changes of Ca2+ might represent an additional control loop. However, as indicated by the question marks, none of these functions has yet been shown experimentally. R, R* and G, G* represent inactive and light-activated rhodopsin and G proteins, respectively. Ca2+ ↑↓ indicates increase or decrease of cytosolic free Ca2+. hγ indicates stimulation of the rhodopsin by blue-green light. Figure 9. Open in new tabDownload slide Model of Possible Functions for Heterotrimeric G Proteins in Photoresponses of Flagellate Green Algae. The model integrates known elements from Chlamydomonas, Haematococcus, and Spermatozopsis. Photoshock is most likely mediated by a pure electrical signal spread toward the flagella, although involvement of G proteins in signaling from the activated rhodopsin to the PR channel under high and low light conditions cannot be completely ruled out (path 1; see Discussion). Possible functions of G proteins downstream of the PR channel are thus likely to be restricted to the phototactic transduction pathway toward the flagella (path 2). Involvement in light adaptation by control of the probability of opening of ion channels in the vicinity of the eyespot apparatus or the PR channel itself (path 3) would be a third intriguing possibility. By stabilizing the lifetime of the activated Gα subunit(s) at increased cytosolic concentrations or enhancing the deactivation at low free cytosolic concentrations, light-induced concentration changes of Ca2+ might represent an additional control loop. However, as indicated by the question marks, none of these functions has yet been shown experimentally. R, R* and G, G* represent inactive and light-activated rhodopsin and G proteins, respectively. Ca2+ ↑↓ indicates increase or decrease of cytosolic free Ca2+. hγ indicates stimulation of the rhodopsin by blue-green light. many systems (e.g., Sweeney and Dolphin, 1992; Li and Assmann, 1993; Clapham, 1994; Armstrong and Blatt, 1995). Thus, regulation of the photoreceptor channel (PR channel) or other voltage-gated ion channels in the eyespot apparatus and its close vicinity to a rhodopsin-coupled G protein would allow a rapid and effective way of light adaptation by control of membrane excitability (Figure 9, path 3), which might also be controlled in a feedback loop by the cytosolic Ca2+ concentration. Here, the observed antagonistic effects of Ca2+ on light modulation of the GTPase(s) offers a neat link to possible functions of the G proteins in adaptational processes. Where might rhodopsin-coupled G proteins additionally be involved in phototactic signaling? As already discussed in detail, direct involvement in signaling from the activated rhodopsin to the PR channel (Figure 9, path 1) cannot be completely ruled out. Because signaling during photoshock can be explained solely by electrical signal spread toward the flagella (reviewed in Hegemann, 1997), additional functions for G proteins are likely to be restricted to the phototactic transduction pathway (Figure 9, path 2). Here, rapid effects of light on the levels of inositol 1,4,5-trisphosphate in Chlamydomonas might be taken as indicative of a phospholipase C as a possible target for a rhodopsin-activated G protein. Illumination of dark-adapted Chlamydomonas with white or photactic active green light leads to a significant increase in the level of inositol 1,4,5-trisphosphate. The maximum is reached within 3 sec (P. Hegemann and G. Mayr, personal communication). Thus, some analogies to many invertebrate systems in which excitation of rhodopsin leads to an increase of inositol 1,4,5-trisphosphate via a Gqα-activated phospholipase C (e.g., Suzuki et al., 1995) might exist. In addition, strong evidence points to the presence of Ca2+-sensitive and G protein–activated phospholipases C and D activities in Chlamydomonas spp and Dunaliella (Einspahr et al., 1989; Yueh and Crain,1993; Munnik et al., 1995). Clearly, more components of the rhodopsin-mediated signaling pathway must be analyzed and defined before we can hope to understand the functions of G proteins in the photoresponses of green algae. However, irrespective of their precise function(s), demonstration of their specific enrichment and light regulation is a first step toward understanding the as yet unknown physiological role(s) of G proteins in green algal photoresponses. METHODS Organism, Culture Conditions, and Membrane Preparations The source, culture of Spermatozopsis similis, and isolation of intact eyespot apparatuses were basically as described by Kreimer et al. (1991a), except that all isolation steps were conducted under dim red light (>650 nm) and the homogenization intervals were reduced to six. To increase yield and structural integrity of the eyespot apparatuses, we diluted fraction 2 twofold with 20 mM Hepes–NaOH, pH 7.8, after the first sucrose gradient centrifugation step and layered directly on the second gradient (10 mL of 20.5% and 10 mL of 31.8% sucrose). Plasma membranes were isolated according to Schlicher et al. (1995). 35S-GTP-γ-S Binding and GTPase Assay Binding of 35S-GTP-γ-S (1 nM; Du Pont–New England Nuclear) to proteins of the eyespot fraction (20 μg) was measured at 4°C, according to Northup et al. (1982), except that the binding and washing buffer contained 2 mM MgCl2, 300 mM NaCl, and 5 mM Tris–HCl, pH 7.5. The reaction was terminated after 10 min by rapid filtration through nitrocellulose filters (HATF 025; Millipore Corp., Bedford, MA). Boiled protein served as a control for nonspecific binding. GTPase activity was determined by following the formation of α-32P-GDP. The assay contained 10 μg of protein, 4 mM MgCl2, 0.5 μM GTP (~6 nM α-32P-GTP; Du Pont–New England Nuclear), 1 μM ATP, 100 μM App(NH)p, and 20 mM Hepes–NaOH, pH 7.5. Assays were conducted either under a red safelight or illumination with the indicated wavelength for 15 to 120 sec. The reaction was terminated by 660 mM sodium formate, pH 3.4. Further processing, thin-layer chromatography separation, and quantification by liquid scintillation counting were basically as described by Millner and Robinson (1989). GTP hydrolysis due to low-affinity GTPases was measured in the presence of 400 μM unlabeled GTP. These values were subtracted from the values for high-affinity GTPase activity. Eyespot apparatuses were not preincubated with the different inhibitors, unless stated otherwise. ADP Ribosylation Activation of Cholera toxin (CTX) and CTX-dependent ADP ribosylation was basically performed as described previously (Gill and Woolkalis, 1991). Briefly, CTX activation was achieved by preincubation in 5 mM DTT, 100 mM NaCl, and 20 mM Hepes–NaOH, pH 7.3 (10 min at 37°C). The final ribosylation assay contained 30 μg of protein, 16 ng of CTX μg−1 of protein, 0.2 mM Gpp(NH)p, 10 mM thymidine, 100 mM NaCl, 2 mM MgCl2, 0.5 mM DTT, 0.5 μM ATP, 100 μM NAD (0.4 μM 32P-NAD), and 20 mM Hepes–NaOH, pH 7.3. The reaction was performed at 20°C and stopped by the addition of 800 μL of ice-cold unlabeled NAD (1 mM). After centrifugation (15 min) in a Beckmann microcentrifuge, the pellets were extracted by using chloroform–methanol–water (4:8:3 [v/v]). Electrophoretic and Other Analyses SDS-PAGE, blotting, immunostaining, and densitometric analysis of autoradiographs were conducted as described previously (Linden and Kreimer, 1995; Schlicher et al., 1995), except that, if not stated otherwise, 11% SDS gels rather than linear gradient gels were used. Gqα peptides were conjugated to BSA, and anti-BSA antibodies were removed by affinity chromatography (Suzuki et al., 1993, 1995). The Giα C-terminal peptide was coupled to keyhole-limpet haemocyanin (Böhm et al., 1993). Isoelectric focusing was performed, with slight modifications, according to O'Farrel (1975). For two-dimensional electrophoresis, minigels were used. For all other analyses, 13 × 17 cm gels were used. Fluoroaluminates (AlF) were formed 1 hr before use by mixing stock solutions of NaF and AlCl3. The final assay concentrations corresponded to 6 mM NaF and 20 μM AlCl3. 3H-retinal binding assay was done basically as described by Beckmann and Hegemann (1991). Fixation of whole cells and processing for electron microscopy were as described by Kreimer et al. (1991a). Living cells were immobilized as described by Kreimer and Melkonian (1990). Safety Light, Illumination, and Filters All experiments for light regulation of the GTPase were done under dim red light (>650 nm, <0.1 μE m−2 sec−1), and exposure of the samples to this light was minimized. A cold light source (Schott, Mainz, Germany) equipped with a dimmer and fiber optics served as the light source in all of our experiments. Illumination was done from the top in a temperature-controlled sample holder. Different quantum fluxes were achieved by using neutral density filters (Schott). Monochromatic filters were from Balzer's (Oberkirch, Germany) and broad-band filters (490 to 570 nm and >560 nm) were from LOT-Oriel (Darmstadt, Germany). ACKNOWLEDGMENTS This study was supported by the Deutsche Forschungsgemeinschaft. We thank Drs. Michael Böhm and Tatsuo Suzuki for kindly providing various Gα antibodies and Dr. Peter Hegemann for the Chlamydomonas rhodopsin antiserum. Special thanks are also given to Drs. Peter Hegemann and Rainer Uhl for sharing information about unpublished work, to Dr. Barbara Surek for critically reading the manuscript, and to Dr. Michael Melkonian for stimulating discussions. We are also grateful to Dr. Andrea Grunow for help with two-dimensional gel electrophoresis. G.K. also thanks the Verein der Freunde und Förderer der Universität zu Köln for support. REFERENCES Armstrong F. Blatt M.R. ( 1995 ). Evidence for K+ channel control in Vicia guard cells coupled by G-proteins to a 7TMS receptor mimetic . Plant J. 8 , 187 – 198 . Google Scholar Crossref Search ADS WorldCat Beck C. ( 1996 ). Lokalisation und Eigenschaften lichtinduzierter Ionenströme in Chlamydomonas reinhardtii . PhD Dissertation ( Munich, Germany : Universität München ). Beck C. Uhl R. ( 1994 ). On the localization of voltage-sensitive calcium channels in the flagella of Chlamydomonas reinhardtii . J. Cell Biol. 125 , 1119 – 1125 . Google Scholar Crossref Search ADS PubMed WorldCat Beckmann M. Hegemann P. ( 1991 ). In vitro identification of rhodopsin in the green alga Chlamydomonas . Biochemistry 30 , 3692 – 3697 . Google Scholar Crossref Search ADS PubMed WorldCat Bentrop J. Paulsen R. ( 1986 ). Light-modulated ADP-ribosylation, protein phosphorylation and protein binding in isolated fly photoreceptor membranes . Eur. J. Biochem. 161 , 61 – 67 . Google Scholar Crossref Search ADS PubMed WorldCat Birnbaumer L. Birnbaumer M. ( 1995 ). G proteins in signal transduction . In Biomembranes: Signal Transduction across Membranes , Vol. 3 , Shinitzky M. , ed ( Weinheim, Germany : VCH–Verlagsgesellschaft ), pp. 153 – 252 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Böhm M. Gräbel C. Kirchmayr R. Lensche H. Erdman E. Gierschik P. ( 1993 ). C-terminal modifications of pertussis toxin-sensitive G-protein α-subunits differentially affect immunoreactivity. Evidence against endogenous ADP-ribosylation in human heart, lung, thrombocytes and adipose tissue . Biochem. Pharmacol. 46 , 2145 – 2154 . Google Scholar Crossref Search ADS PubMed WorldCat Clapham D.E. ( 1994 ). Direct G protein activation of ion channels? Annu. Rev. Neurosci. 17 , 441 – 464 . Google Scholar Crossref Search ADS PubMed WorldCat Deininger W. Kröger P. Hegemann U. Lottspeich F. Hegemann P. ( 1995 ). Chlamyrhodopsin represents a new type of sensory photoreceptor . EMBO J. 4 , 5849 – 5858 . Google Scholar Crossref Search ADS WorldCat Dumler I.L. Korolkov S.N. Garnovskaya M.N. Parfenova D.V. Etinghof R.N. ( 1989 ). The systems of photo- and pheromone signal transduction in unicellular eukaryotes . J. Protein Chem. 8 , 387 – 389 . Google Scholar Crossref Search ADS PubMed WorldCat Einspahr K.J. Peeler T.C. Thompson G.A. Jr. ( 1989 ). Phosphatidylinositol 4,5-bisphosphate phospholipase C and phosphomonoesterase in Dunaliella salina membranes . Plant Physiol. 90 , 1115 – 1120 . Google Scholar Crossref Search ADS PubMed WorldCat Foster K.W. Smyth R.D. ( 1980 ). Light antennas in phototactic algae . Microbiol. Rev. 44 , 572 – 630 . Google Scholar Crossref Search ADS PubMed WorldCat Foster K.W. Saranak J. Patel N. Zarilli G. Okabe M. Kline T. Nakanishi K. ( 1984 ). A rhodopsin is the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas . Nature 311 , 756 – 759 . Google Scholar Crossref Search ADS PubMed WorldCat Foster K.W. Saranak J. Zarilli G. ( 1988 ). Autoregulation of rhodopsin synthesis in Chlamydomonas reinhardtii . Proc. Natl. Acad. Sci. USA 85 , 6379 – 6383 . Google Scholar Crossref Search ADS WorldCat Fyles J.M. Baverstock J. Baer K. Saibil H.R. ( 1991 ). Effects of calcium on light-activated GTP-binding proteins in squid photoreceptor membranes . Comp. Biochem. Physiol. 98B , 215 – 221 . Google Scholar OpenURL Placeholder Text WorldCat Gärtner W. Towner P. ( 1995 ). Invertebrate pigments and chromophore protein interactions . Photochem. Photobiol. 62 , 1 – 16 . Google Scholar Crossref Search ADS PubMed WorldCat Gill D.M. Woolkalis M.J. ( 1991 ). Cholera toxin-catalyzed [32P]ADP-ribosylation of proteins . Methods Enzymol. 195 , 267 – 280 . Google Scholar Crossref Search ADS PubMed WorldCat Gotor C. Lam E. Cejudo F.J. Romero L.C. ( 1996 ). Isolation and analysis of the soybean SGA2 gene (cDNA), encoding a new member of the plant G-protein family of signal transducers . Plant Mol. Biol. 32 , 1227 – 1234 . Google Scholar Crossref Search ADS PubMed WorldCat Grand R.J.A. Owen D. ( 1991 ). The biochemistry of ras p21 . Biochem. J. 279 , 609 – 631 . Google Scholar Crossref Search ADS PubMed WorldCat Harz H. Hegemann P. ( 1991 ). Rhodopsin-regulated calcium currents in Chlamydomonas . Nature 351 , 489 – 491 . Google Scholar Crossref Search ADS WorldCat Harz H. Nonnengässer C. Hegemann P. ( 1992 ). The photoreceptor current of the green alga Chlamydomonas . Philos. Trans. R. Soc. Lond. B 338 , 39 – 52 . Google Scholar Crossref Search ADS WorldCat Hegemann P. ( 1997 ). Vision in microalgae . Planta 203 , 265 – 274 . Google Scholar Crossref Search ADS PubMed WorldCat Hegemann P. Harz H. ( 1993 ). Photoreception in Chlamydomonas . In Signal Transduction: Prokaryotic and Simple Eukaryotic Systems , Kurjan J. Taylor B.L. , eds ( San Diego, CA : Academic Press ), pp. 279 – 307 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Horst C.J. Witman G.B. ( 1993 ). ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance . J. Cell Biol. 120 , 733 – 741 . Google Scholar Crossref Search ADS PubMed WorldCat Kahn R.A. ( 1991 ). Fluoride is not an activator of the smaller (20–25 kD) GTP-binding proteins . J. Biol. Chem. 266 , 15595 – 15597 . Google Scholar Crossref Search ADS PubMed WorldCat Koelle M.R. ( 1997 ). A new family of G-protein regulators—The RGS proteins . Curr. Opin. Cell Biol. 9 , 143 – 147 . Google Scholar Crossref Search ADS PubMed WorldCat Korolkov S.N. Garnovskaya M.N. Basov A.S. Chuanev A.S. Dumler I.L. ( 1990 ). The detection and characterization of G-proteins in the eyespot of Chlamydomonas reinhardtii . FEBS Lett. 270 , 132 – 134 . Google Scholar Crossref Search ADS PubMed WorldCat Kreimer G. ( 1994 ). Cell biology of phototaxis in flagellate algae . Int. Rev. Cytol. 148 , 229 – 310 . Google Scholar Crossref Search ADS WorldCat Kreimer G. Melkonian M. ( 1990 ). Reflection confocal laser scanning microscopy of eyespots in flagellated green algae . Eur. J. Cell Biol. 53 , 101 – 111 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Kreimer G. Brohsonn U. Melkonian M. ( 1991a ). Isolation and partial characterization of the photoreceptive organelle for phototaxis of a flagellate green alga . Eur. J. Cell Biol. 55 , 318 – 327 . Google Scholar OpenURL Placeholder Text WorldCat Kreimer G. Marner F.-J. Brohsonn U. Melkonian M. ( 1991b ). Identification of 11-cis and all-trans-retinal in the photoreceptive organelle of a flagellate green alga . FEBS Lett. 293 , 49 – 52 . Google Scholar Crossref Search ADS WorldCat Kröger P. Hegemann P. ( 1994 ). Photophobic responses and phototaxis in Chlamydomonas are triggered by a single rhodopsin photoreceptor . FEBS Lett. 341 , 5 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat Li W. Assmann S.M. ( 1993 ). Characterization of a G-protein–regulated outward K+ current in mesophyll cells of Vicia faba L . Proc. Natl. Acad. Sci. USA 90 , 262 – 266 . Google Scholar Crossref Search ADS WorldCat Linden L. Kreimer G. ( 1995 ). Calcium modulates rapid protein phosphorylation/dephosphorylation in isolated eyespot apparatuses of the green alga Spermatozopsis similis . Planta 197 , 343 – 351 . Google Scholar Crossref Search ADS WorldCat Litvin F.F. Sineshchekov O.A. Sineschekov V.A. ( 1978 ). Photoreceptor electric potential in the phototaxis of the alga Haematococcus pluvialis . Nature 271 , 476 – 478 . Google Scholar Crossref Search ADS PubMed WorldCat Ma H. ( 1994 ). GTP-binding proteins in plants: New members of an old family . Plant Mol. Biol. 26 , 1611 – 1636 . Google Scholar Crossref Search ADS PubMed WorldCat McFadden G.I. Schulze D. Surek B. Salisbury J.L. Melkonian M. ( 1987 ). Basal body reorientation mediated by a Ca2+-modulated contractile protein . J. Cell Biol. 105 , 903 – 912 . Google Scholar Crossref Search ADS PubMed WorldCat Melkonian M. Robenek H. ( 1984 ). The eyespot apparatus of flagellated green algae . Prog. Phycol. Res. 3 , 193 – 268 . Google Scholar OpenURL Placeholder Text WorldCat Millar A.J. McGrath R.B. Chua N.-H. ( 1994 ). Phytochrome phototransduction pathways . Annu. Rev. Genet. 28 , 325 – 349 . Google Scholar Crossref Search ADS PubMed WorldCat Millner P.A. Causier B.E. ( 1996 ). G-protein coupled receptors in plant cells . J. Exp. Bot. 47 , 983 – 992 . Google Scholar Crossref Search ADS WorldCat Millner P.A. Robinson P.S. ( 1989 ). ADP-ribosylation of thylakoid membrane polypeptides by cholera toxin is correlated with inhibition of thylakoid GTPase activity and protein phosphorylation . Cell. Signalling 1 , 421 – 433 . Google Scholar Crossref Search ADS WorldCat Morel-Laurens N. ( 1987 ). Calcium control of phototactic orientation in Chlamydomonas reinhardtii: Sign and strength of response . Photochem. Photobiol. 45 , 119 – 128 . Google Scholar Crossref Search ADS PubMed WorldCat Munnik T. Arisz S.A. de Vrije T. Musgrave A. ( 1995 ). G protein activation stimulates phospholipase D signaling in plants . Plant Cell 7 , 2197 – 2210 . Google Scholar Crossref Search ADS PubMed WorldCat Nonnengässer C. Holland E.-M. Harz H. Hegemann P. ( 1996 ). The nature of rhodopsin-triggered photocurrents in Chlamydomonas. II. Influence of monovalent ions . Biophys. J. 70 , 932 – 938 . Google Scholar Crossref Search ADS PubMed WorldCat Northup J.K. Smigel M.D. Gilman A.G. ( 1982 ). The guanine nucleotide activating site of the regulatory component of adenylate cyclase . J. Biol. Chem. 257 , 11416 – 11423 . Google Scholar Crossref Search ADS PubMed WorldCat O'Farrel P.H. ( 1975 ). High resolution two-dimensional electrophoresis of proteins . J. Biol. Chem. 250 , 4007 – 4040 . Google Scholar Crossref Search ADS PubMed WorldCat Pazour G.J. Sineshchekov O.A. Witman G.B. ( 1995 ). Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii . J. Cell Biol. 131 , 427 – 440 . Google Scholar Crossref Search ADS PubMed WorldCat Pennington S.R. ( 1994 ). GTP-binding proteins: Heterotrimeric G proteins . Protein Profile 1 , 169 – 342 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Preisig H.R. Melkonian M. ( 1984 ). A light and electron microscopical study of the green flagellate Spermatozopsis similis spec . nova. Plant Syst. Evol. 146 , 57 – 74 . Google Scholar Crossref Search ADS WorldCat Schlicher U. Linden L. Calenberg M. Kreimer G. ( 1995 ). G proteins and Ca2+-modulated protein kinases of a plasma membrane–enriched fraction and isolated eyespot apparatuses of Spermatozopsis similis (Chlorophyceae) . Eur. J. Phycol. 30 , 319 – 330 . Google Scholar Crossref Search ADS WorldCat Schloss J.A. ( 1990 ). A Chlamydomonas gene encodes a G protein β subunit–like polypeptide . Mol. Gen. Genet. 221 , 443 – 452 . Google Scholar Crossref Search ADS PubMed WorldCat Schumaker K. Gizinski M.J. ( 1996 ). G proteins regulate dihydropyridine binding to moss plasma membranes . J. Biol. Chem. 271 , 21292 – 21296 . Google Scholar Crossref Search ADS PubMed WorldCat Sineshchekov O.A. Litvin F.F. Keszthelyi L. ( 1990 ). Two components of photoreceptor potential in phototaxis of the flagellated green alga Haematococcus pluvialis . Biophys. J. 57 , 33 – 39 . Google Scholar Crossref Search ADS PubMed WorldCat Suzuki T. Narita K. Yoshihara K. Nagai K. Kito Y. ( 1993 ). Immunochemical detection of GTP-binding protein in cephalopod photoreceptors by anti-peptide antibodies . Zool. Sci. 10 , 425 – 430 . Google Scholar OpenURL Placeholder Text WorldCat Suzuki T. Narita K. Yoshihara K. Nagai K. Kito Y. ( 1995 ). Phosphatidyl inositol-phospholipase C in squid photoreceptor membrane is activated by stable metarhodopsin via GTP-binding protein, Gq . Vision Res. 35 , 1011 – 1017 . Google Scholar Crossref Search ADS PubMed WorldCat Sweeney M.I. Dolphin A.C. ( 1992 ). 1,4-Dihydropyridines modulate GTP hydrolysis by G0 in neuronal membranes . FEBS Lett. 310 , 66 – 70 . Google Scholar Crossref Search ADS PubMed WorldCat Terryn N. Van Montagu M. Inzé D. ( 1993 ). GTP-binding proteins in plants . Plant Mol. Biol. 22 , 143 – 152 . Google Scholar Crossref Search ADS PubMed WorldCat Torres-Márquez M.E. Macias-Silva M. Vega-Segura A. ( 1996 ). Identification of a functional Gs protein in Euglena gracilis . Comp. Biochem. Physiol. 115C , 233 – 237 . Google Scholar OpenURL Placeholder Text WorldCat von Kampen J. Nieländer U. Wettern M. ( 1994 ). Stress-dependent transcription of a gene encoding a Gβ-like polypeptide from Chlamydomonas reinhardtii . J. Plant Physiol. 143 , 756 – 758 . Google Scholar Crossref Search ADS WorldCat Warpeha K.M.F. Hamm H.E. Rasenick M.M. Kaufman L.S. ( 1991 ). A blue-light–activated GTP-binding protein in the plasma membrane of etiolated peas . Proc. Natl. Acad. Sci. USA 88 , 8925 – 8929 . Google Scholar Crossref Search ADS WorldCat Weiss C.A. White E. Huang H. Ma H. ( 1997 ). The G protein α subunit (GPα1) is associated with the ER and the plasma membrane in meristematic cells of Arabidopsis and cauliflower . FEBS Lett. 407 , 361 – 367 . Google Scholar Crossref Search ADS PubMed WorldCat Witman G.B. ( 1993 ). Chlamydomonas phototaxis . Trends Cell Biol. 3 , 403 – 408 . Google Scholar Crossref Search ADS PubMed WorldCat Wu Y. Hiratsuka K. Neuhaus G. Chua N.-H. ( 1996 ). Calcium and cGMP target distinct phytochrome-responsive elements . Plant J. 10 , 1149 – 1154 . Google Scholar Crossref Search ADS PubMed WorldCat Yueh Y.G. Crain R.C. ( 1993 ). Deflagellation of Chlamydomonas reinhardtii follows a rapid transitory accumulation of inositol 1,4,5,-trisphosphate and requires Ca2+ entry . J. Cell Biol. 123 , 869 – 875 . Google Scholar Crossref Search ADS PubMed WorldCat Zacks D.N. Spudich J.L. ( 1994 ). Gain setting in Chlamydomonas reinhardtii: Mechanism of phototaxis and the role of the photophobic response . Cell Motil. Cytoskel. 29 , 225 – 230 . Google Scholar Crossref Search ADS WorldCat Author notes 1 To whom correspondence should be addressed. E-mail gkreimer@novell.biolan.uni-koeln.de; fax 49-221-470-5181. © 1998 American Society of Plant Physiologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Light- and Ca2+-Modulated Heterotrimeric GTPases in the Eyespot Apparatus of a Flagellate Green Alga
JF - The Plant Cell
DO - 10.1105/tpc.10.1.91
DA - 1998-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/light-and-ca2-modulated-heterotrimeric-gtpases-in-the-eyespot-dyUkvwfpWI
SP - 91
EP - 103
VL - 10
IS - 1
DP - DeepDyve
ER -