Minisymposium 14: Protein Modification

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Abs # M1404: Analysis of lipid modifications of a type-I ROP by GC/MS reveals coordinate regulation by GDP/GTP exchange and transient S-acylation

Presenter:	Yalovsky, Shaul       Contact Presenter
Authors	Yalovsky, Shaul  (A)   Sorek, Nadav  (A)   Poraty, Limor  (A)   Sternberg, Hasana  (A)   Bar, Enat  (B)   Lewinsohn, Efraim  (B)
Affiliations:	(A): Department of Plant Sciences, Tel Aviv University (B): Department of Field and Vegetable Crops, Agricultural Research Organization, Newe Yaar Reseach Center
Web Site:	http://www.tau.ac.il/lifesci/departments/plant_s/members/yalovsky/yalovsky.html

ROPs are plant Rac-related GTPases that are attached to the membrane by virtue of posttranslational lipid modifications. Regardless of their importance for function, the exact identities of ROPs' lipid modifications had not been established, nor were the connections between activation status of these GTPases, their lipid modifications and distribution in membrane microdomains. Using cell fractionations by centrifugation and membrane floatation assays in conjunction with protein immunoblots and gas chromatography coupled mass spectrometry (GC/MS) a connection between the activation status of ROPs, their posttranslational lipid modifications and distribution in the membrane has been established. Protein immunoblots with affinity-purified anti-ROP Abs demonstrated that ROPs were exclusively membrane localized, distributed between non-ionic detergent soluble and Detergent Resistant Membranes (DRMs). Introduction of the non-hydrolysable GTP analog, GTPγS, into leaves or seedlings induced ROPs' accumulation in DRMs, while introduction of GDP induced accumulation in detergent soluble membrane fractions. Wild type and activated forms of the type-I ROP, AtROP6, were ectopically expressed in Arabidopsis. The WT AtROP6 was dispersed between detergent soluble and DRMs while the activated AtROP6 accumulated exclusively in DRMs. The recombinant proteins were purified from plants and in turn lipids were extracted from them and analyzed by GC/MS. This analysis showed that AtROP6 purified from detergent soluble membranes was only prenylated while the proteins that accumulated in DRMs were both prenylated and S-acylated. The major modifying prenyl group was geranylgeranyl but low levels of farnesyl were also detected. AtROP6 was S-acylated by both palmitic and stearic acids. A prenylation compromised AtROP6 mutant transgene was also not S-acylated, suggesting that in vivo prenylation precedes S-acylation. The results indicate that GTP binding and activation of type-I ROPs induces their S-acylation and accumulation in DRMs. In turn, GTP hydrolysis induces loss of the acyl groups and consequent redistribution of the ROPs in detergent soluble membranes. Our findings demonstrate that GDP/GTP exchange and transient S-acylation synergistically regulate ROPs' signaling.