Testing the Phenotypic Effects of a Rab Chimera that Resolves Exchange Factor Specificity from Effector Specificity.

Abstract

Rab GTPases play key roles in defining the identity of the various compartments that comprise the secretory and endocytic pathways. Recruitment of a Rab to a specific compartment requires its localized activation by a guanine nucleotide exchange factor (GEF). This in turn results in the recruitment of a distinct set of Rab effectors that directs the recognition of the appropriate target compartment by a carrier vesicle and their subsequent fusion. A chimeric Rab protein, Ypt1-SW1Sec4, was found to separate GEF specificity from effector specificity (Grosshans BL, et al. Proc Natl Acad Sci U S A 103(32):11821-11827, 2006), but early studies did not observe strong effects of this allele on growth or membrane traffic (Brennwald P, Novick P. Nature 362(6420):560-563, 1993). To resolve this apparent conundrum, yeast strains expressing the chimeric Rab were subjected to a more extensive battery of phenotypic tests. These tests demonstrated that changing the specificity of the GEF interaction does lead to a change in Rab localization and can lead to the ectopic recruitment of an effector, creating trafficking defects that are dependent upon the level of expression (Grosshans BL, et al. Proc Natl Acad Sci U S A 103(32):11821-11827, 2006). Here we describe the methods used in this analysis. Specifically we describe the following: 1. An assay used to quantify the efficiency of export of a cell wall protein Bgl2, 2. The use of thin section electron microscopy to address the morphology of the secretory machinery, 3. The use of a fluorescently tagged vesicle SNARE protein, GFP-Snc1, to follow plasma membrane recycling and. 4. The use of fluorescently tagged Ypt1 effectors, Cog3-GFP, Uso1-GFP, and Sec7-GFP to follow their recruitment by Ypt1-SW1Sec4.