Differential Effects of Prenylation and S-Acylation
on Type I and II ROPS Membrane Interaction
, Orit Gutman, Einat Bar, Mohamad Abu-Abied, Xuehui Feng, Mark P. Running,
Efraim Lewinsohn, Naomi Ori, Einat Sadot, Yoav I. Henis, and Shaul Yalovsky*
Department of Molecular Biology and Ecology of Plants (N.S., S.Y.) and Department of Neurobiology (O.G.,
Y.I.H.), George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel; Donald Danforth
Plant Science Center, St. Louis, Missouri 63132 (X.F., M.P.R.); Department of Field and Vegetable Crops,
Agricultural Research Organization, Neve Ya’ar Research Center, Ramat Yishay 30095, Israel (E.B., E.L.);
Robert Smith Institute of Plant Sciences and Genetics of Agriculture, Faculty of Agriculture, Hebrew
University of Jerusalem, Rehovot 76100, Israel (N.O.); and Department of Ornamental Horticulture,
Agricultural Research Organization, Volcani Center, Bet-Dagan 50250, Israel (M.A.-A., E.S.)
Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs)
and Gg proteins, while type II ROPs are attached to the plasma membrane by S-acylation. Yet, it is not known how prenylation
affects ROP membrane interaction dynamics and what are the functional redundancy and speciﬁcity of type I and type II
ROPs. Here, we have used the expression of ROPs in mammalian cells together with geranylgeranylation and CaaX
prenylation-deﬁcient mutants to answer these questions. Our results show that the mechanism of type II ROP S-acylation and
membrane attachment is unique to plants and likely responsible for the viability of plants in the absence of CaaX prenylation
activity. The prenylation of ROPs determines their steady-state distribution between the plasma membrane and the cytosol but
has little effect on membrane interaction dynamics. In addition, the prenyl group type has only minor effects on ROP function.
Phenotypic analysis of the CaaX prenylation-deﬁcient pluripetala mutant epidermal cells revealed that type I ROPs affect cell
structure primarily on the adaxial side, while type II ROPs are functional and induce a novel cell division phenotype in this
genetic background. Taken together, our studies show how prenyl and S-acyl lipid modiﬁcations affect ROP subcellular
distribution, membrane interaction dynamics, and function.
Protein prenylation involves the covalent attach-
ment of the C15 and C20 isoprenoids farnesyldiphos-
phate (FPP) and geranylgeranyldiphosphate (GGPP)
to Cys residues in the C-terminal CaaX box or in
C-terminal double Cys motifs of Rab small G proteins.
Prenylation is required for membrane targeting and
function of diverse protein groups, many of which
have key regulatory functions (Maurer-Stroh et al.,
2003, 2007; Magee and Seabra, 2005; Crowell and
Huizinga, 2009; Sorek et al., 2009). Prenylation of
CaaX box proteins is catalyzed by two distinct prenyl-
transferases: protein farnesyltransferase (PFT) and
protein geranylgeranyltransferase-I (PGGT-I; Maurer-
Stroh et al., 2003). PFT and PGGT-I are heterodimeric
proteins composed of a common a-subunit and dis-
tinct substrate-speciﬁc b-subunits (Maurer-Stroh et al.,
2003). Both PFT and PGGT-I are conserved in plants
(Yalovsky et al., 1997; Caldelari et al., 2001). PFT and
PGGT-I recognize a conserved C-terminal sequence
known as the CaaX box, in which C is a Cys, a usually
represents an aliphatic amino acid, and X is usually
Ser, Met, Cys, Ala, Gln, or Leu. If X is a Leu, the protein
is geranylgeranylated by PGGT-I. If X is another amino
acid, the protein is preferentially farnesylated by PFT
(Reiss et al., 1991; Seabra et al., 1991; Maurer-Stroh
et al., 2003; Reid et al., 2004). The presence of an Arg/
Lys-rich polybasic domain proximal to the CaaX box
greatly increases substrate afﬁnity of PGGT-I (James
et al., 1995; Caldelari et al., 2001).
PFT and PGGT-I are in part promiscuous and
PGGT-I can prenylate PFT substrates, albeit inefﬁ-
ciently (Trueblood et al., 1993; Armstrong et al., 1995;
This work was supported by the Israel Science Foundation
(grant no. ISF–312/07 to S.Y.), the United States-Israel Binational
Research and Development Fund (grant no. BARD–IS–4032–07 to
S.Y.), the Deutschland-Israel Program (grant no. DIP–H.3.1 to S.Y.),
and by an Eshkol Fellowship for Ph.D. students from the Israel
Ministry of Science and Technology to N.S. Y.I.H. is an incumbent of
the Zalman Weinberg Chair in Cell Biology.
Present address: Energy Biosciences Institute, University of
California, Berkeley, CA 94720.
* Corresponding author; e-mail email@example.com.
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.plantphysiol.org) is:
Shaul Yalovsky (firstname.lastname@example.org).
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706 Plant Physiology
, February 2011, Vol. 155, pp. 706–720, www.plantphysiol.org Ó 2010 American Society of Plant Biologists