An S-acylation switch of conserved G domain cysteines is required for polarity signaling by ROP GTPases.
Rho GTPases are master regulators of cell polarity. For their function, Rhos must associate with discrete plasma membrane domains. Rho of Plants (ROPs) or RACs comprise a single family. Prenylation and S-acylation of hypervariable domain cysteines of Ras and Rho GTPases are required for their function; however, lipid modifications in the G domain have never been reported. Reversible S-acylation involves the attachment of palmitate (C16:0) or other saturated lipids to cysteines through a thioester linkage and was implicated in the regulation of signaling. Here we show that transient S-acylation of Arabidopsis AtROP6 takes place on two conserved G domain cysteine residues, C21 and C156. C21 is relatively exposed and is accessible for modification, but C156 is not, implying that its S-acylation involves a conformational change. Fluorescence recovery after photobleaching beam-size analysis shows that S-acylation of AtROP6 regulates its membrane-association dynamics, and detergent-solubilization studies indicate that it regulates AtROP6 association with lipid rafts. Site-specific acylation-deficient AtROP6 mutants can bind and hydrolyze GTP but display compromised effects on polar cell growth, endocytic uptake of the tracer dye FM4-64, and distribution of reactive oxygen species. These data reveal an S-acylation switch that regulates Rho signaling.
Sorek
Nadav
N
Department of Molecular Biology and Ecology of Plants, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Segev
Oshik
O
Gutman
Orit
O
Bar
Einat
E
Richter
Sandra
S
Poraty
Limor
L
Hirsch
Joel A
JA
Henis
Yoav I
YI
Lewinsohn
Efraim
E
Jürgens
Gerd
G
Yalovsky
Shaul
S
D000215:Acylation; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology / Q000502:physiology; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D016764:Cell Polarity; D003545:Cysteine / Q000378:metabolism*; D050356:Lipid Metabolism; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013229:Stearic Acids / Q000378:metabolism
D029681:Arabidopsis Proteins; C518506:ROP6 protein, Arabidopsis; D011993:Recombinant Fusion Proteins; D013229:Stearic Acids; D019308:Palmitic Acid; C031183:stearic acid; D020559:Monomeric GTP-Binding Proteins; D003545:Cysteine