Activation status-coupled transient S acylation determines membrane partitioning of a plant Rho-related GTPase.
ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane by virtue of posttranslational lipid modifications. The relationship between ROP activation status and membrane localization has not been established. Here we demonstrate that endogenous ROPs, as well as a transgenic His(6)-green fluorescent protein (GFP)-AtROP6 fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, an activated His(6)-GFP-Atrop6(CA) mutant protein accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPgammaS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 isoforms were purified from Arabidopsis plants, and their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids. The acyl lipids were identified as palmitic and stearic acids. In agreement, activated His(6)-GFP-Atrop6(CA)mS(156) in which cysteine(156) was mutated into serine accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6 and possibly other ROPs are transiently S acylated, which induces their partitioning into detergent-resistant membranes.
Sorek
Nadav
N
Department of Plant Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.
Poraty
Limor
L
Sternberg
Hasana
H
Bar
Enat
E
Lewinsohn
Efraim
E
Yalovsky
Shaul
S
D000215:Acylation; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation; D008401:Gas Chromatography-Mass Spectrometry; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017368:Protein Prenylation; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D029681:Arabidopsis Proteins; D003902:Detergents; D008055:Lipids; C518506:ROP6 protein, Arabidopsis; D020559:Monomeric GTP-Binding Proteins; D020741:rho GTP-Binding Proteins