Characterization of PRA2 and VAP-A, two regulators of protein trafficking between the endoplasmic reticulum and Golgi compartments

Abstract

Regulation of vesicle trafficking is required to maintain the specific make-up of each membrane compartment. Prenylated Rab acceptor 1 (PRA1) is known to interact with Rab GTPases and VAMP2, proteins involved in vesicle trafficking, suggesting PRA isoforms could regulate vesicle trafficking. The first aim was to characterize PRA's interaction with Rabs and VAMP2. Using point mutations and in vitro binding assays, I discovered that PRAs interact with multiple Rabs independent of their guanine nucleotide-bound state and identified three residues (N70, S76, H166), whose mutation induced a strong modification to PRA1's interaction with both Rab3A and VAMP2. These results suggest PRA might employ the same binding site to interact with multiple proteins. The second aim was to identify novel PRA-interacting proteins and clarify the function of PRAs. I initiated a yeast two-hybrid screen with PRA1 as bait, and identified VAMP-associated protein A (VAP-A), a protein also believed to be involved in vesicle trafficking. In CHO cells, VAP-A extensively co-localized with the ER-localized PRA2 rather than the predominantly Golgi-localized PRA1, and co-immunoprecipitation confirmed the in vivo PRA2-VAP-A interaction. In immunofluorescence and biochemical assays, overexpression of VAP-A or PRA2 delayed the ER-to-Golgi trafficking of the secretory pathway marker VSVGts045. Also, FRAP assays demonstrated that unlike PRA2, VAP-A achieved this by inhibiting the ER-lateral movement of VSVG ts045, thereby delaying the protein's exit from the ER. ER budding assays confirmed that VAP-A overexpression reduced VSVGts045 sequestration onto vesicles. The addition of the microtubule-depolymerizing agent nocodazole rescued the VAP-A-induced reduction in VSVGts045 budding. This suggests that VAP-A creates static subcompartments of the ER via its interaction with microtubules preventing the lateral movement of VSVG ts045. Overexpression of PRA2 also reduced the levels of VSVG ts045 found on vesicles collected in the budding assay, and unexpectedly, microtubule depolymerization resulted in a doubling of VSVGts045 release from the ER. This suggests PRA2 overexpression has two effects: PRA2 can increase vesicle formation from the ER, but only when its dominant microtubule-dependent inhibitory effect is alleviated by microtubule depolymerization. The results I obtained suggest that both PRA2 and VAP-A delay the ER-to-Golgi trafficking of VSVGts045, but achieve this through different mechanisms.