Chlamydomonas reinhardtii Has Multiple Prolyl
4-Hydroxylases, One of Which Is Essential for Proper
Cell Wall Assembly
and Johanna Myllyharju
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu,
FIN-90014 Oulu, Finland
Biocenter Oulu and Department of Pathology, University of Oulu, FIN-90014 Oulu, Finland
Prolyl 4-hydroxylases (P4Hs) catalyze formation of 4-hydroxyproline (4Hyp), which is found in many plant glycoproteins. We
cloned and characterized Cr-P4H-1, one of 10 P4H-like Chlamydomonas reinhardtii polypeptides. Recombinant Cr-P4H-1 is a
soluble 29-kD monomer that effectively hydroxylated in vitro both poly(
-Pro) and synthetic peptides representing Pro-rich
motifs found in the Chlamydomonas cell wall Hyp-rich glycoprotein (HRGP) GP1. Similar Pro-rich repeats that are likely to be
Cr-P4H-1 substrates are also present in the cell wall HRGP GP2 and probably GP3. Suppression of the gene encoding Cr-P4H-1
by RNA interference led to a defective cell wall consisting of a loose network of ﬁbrils resembling the inner and outer W1 and W7
layers of the wild-type wall, while the layers forming the dense central triplet were absent. The lack of Cr-P4H-1 most probably
affected 4Hyp content of the major HRPGs of the central triplet, GP1, GP2, and GP3. The reduced 4Hyp levels in these HRGPs
can also be expected to affect their glycosylation and, thus, the interactive properties and stabilities of their ﬁbrous shafts.
Interestingly, our RNA interference data indicate that the nine other Chlamydomonas P4H-like polypeptides could not fully
compensate for the lack of Cr-P4H-1 activity and are therefore likely to have different substrate speciﬁcities and functions.
4-Hydroxyproline (4Hyp) is found in many Hyp-rich plant glyco-
proteins (HRGPs) that are the major structural components of cell
walls (for reviews, see Cassab, 1998; Kieliszewski and Shpak,
2001). The HRGP family contains three major subgroups: repet-
itive Pro-rich proteins, extensins, and arabinogalactan proteins
(Cassab, 1998; Kieliszewski and Shpak, 2001). The cell walls in
green algae are almost entirely built up from extensin-like HRGPs,
the Chlamydomonas reinhardtii cell wall containing 25 to 30 dif-
ferent HRGPs (Adair and Snell, 1990; Sumper and Hallmann,
1998). In addition, sexual adhesion of C. reinhardtii gametes
during mating occurs via agglutinins that are also HRGPs (Ferris
et al., 2005). Three classes of Pro-rich motifs that consist of con-
tiguous Pro residues, Pro residues alternating with some other
amino acid, mostly Ser, and -Pro-Pro-Ser-Pro-X- repeats have
been identiﬁed in algal HRGPs, with many of the Pro residues
being subsequently 4-hydroxylated (Adair and Snell, 1990; Sumper
and Hallmann, 1998; Ferris et al., 2001).
Most of the 4Hyp in animal proteins is found in collagens and
>20 additional proteins with collagen-like sequences. 4Hyp res-
idues play a critical role in all collagens, as they are essential for
the formation of stable collagen triple helices at body tempera-
ture (for reviews, see Kivirikko and Pihlajaniemi, 1998; Myllyharju,
2003; Myllyharju and Kivirikko, 2004).
The formation of 4Hyp in all these proteins is catalyzed by
prolyl 4-hydroxylases (P4Hs), which act on Pro residues in pep-
tide linkages and require Fe
, 2-oxoglutarate, O
, and ascorbate
(Kivirikko and Pihlajaniemi, 1998; Myllyharju, 2003). The well-
characterized vertebrate type I, II, and III collagen P4Hs (C-P4Hs)
reside within the lumen of the endoplasmic reticulum and are
tetramers in which the catalytic a subunits have three iso-
forms: a(I), a(II), and a(III) (Helaakoski et al., 1989; Annunen et al.,
1997; Kukkola et al., 2003; Van Den Diepstraten et al., 2003).
Animal C-P4Hs have also been identiﬁed and characterized from
nematodes (Veijola et al., 1994; Friedman et al., 2000; Winter
and Page, 2000; Merriweather et al., 2001; Myllyharju et al.,
2002; Riihimaa et al., 2002; Winter et al., 2003) and Drosophila
melanogaster (Annunen et al., 1999; Abrams and Andrew, 2002).
A second family of animal P4Hs has a cytoplasmic and nuclear
location. It consists of three isoenzymes with no b subunit that
play a central role in the oxygen-dependent regulation of the
hypoxia-inducible transcription factor HIF (Bruick and McKnight,
2001; Epstein et al., 2001; Ivan et al., 2002). These HIF-P4Hs are
effective oxygen sensors due to their high K
values for O
et al., 2003).
Plant P4Hs have been partially puriﬁed and characterized from
many higher plant sources (for a review, see Kivirikko et al., 1992)
and the green algae C. reinhardtii and Volvox carteri (Kaska et al.,
1987, 1988), but the only plant P4Hs cloned and characterized so
far are two monomeric enzymes, At-P4Hs 1 and 2, from Arabi-
dopsis thaliana and one from Nicotiana tabacum (Hieta and
To whom correspondence should be addressed. E-mail johanna.
myllyharju@oulu.ﬁ; fax 358-8-537 5811.
The author responsible for distribution of materials integral to the
ﬁndings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Johanna Myllyharju
Online version contains Web-only data.
The Plant Cell, Vol. 19: 256–269, January 2007, www.plantcell.org
2007 American Society of Plant Biologists