The GTPase-Activating Enzyme Gyp1p Is Required for Recycling of Internalized Membrane Material by Inactivation of the Rab/Ypt GTPase Ypt1p

Abstract

Rab/Ypt GTPases are key regulators of membrane trafficking and together with SNARE proteins mediate selective fusion of vesicles with target compartments. A family of GTPase-activating enzymes (GAPs) specific for Rab/Ypt GTPases has been discovered, but little is known about their function and substrate specificity in vivo. Here we show that the GAP activity of Gyp1p, a yeast member of this family, is specifically required for recycling of the SNARE Snc1p and the membrane dye FM4-64, implying that inactivation of a Rab/Ypt GTPase may be necessary for recycling of membrane material. Interestingly, recycling of GFP-Snc1p in gyp1⌬ cells is partially restored by reducing the activity of Ypt1p. Moreover, GFP-Snc1p accumulated intracellularly in wild-type cells expressing a GTP-locked, mutant form of Ypt1p (Ypt1p-Q67L), suggesting that GTP hydrolysis of Ypt1p is essential for recycling. Ypt6p is known to be required for the fusion of recycling vesicles to the late Golgi compartment. Interestingly, the deletions of GYP1 and YPT6 were synthetic lethal, raising the possibility that at least two distinct pathways are involved in recycling of membrane material.

A Rab GAP cascade defines the boundary between two Rab GTPases on the secretory pathway

“…The CPY mis-sorting defect is most likely an indirect effect of a block in recycling of the CPY receptor Vps10p from the endosome back to the Golgi (14). Synthetic lethality has been observed between gyp1⌬ and a large number of other mutations, including many that affect components of the Golgi apparatus (13,14). Thus, the disorganization of the Golgi that results from the loss of Gyp1p antagonizes mutants already deficient in some other aspect of Golgi function.…”

“…This shift in COG localization may affect the recycling to the cell surface. The CPY mis-sorting defect is most likely an indirect effect of a block in recycling of the CPY receptor Vps10p from the endosome back to the Golgi (14). Synthetic lethality has been observed between gyp1⌬ and a large number of other mutations, including many that affect components of the Golgi apparatus (13,14).…”

“…The loss of Gyp1p function results in several trafficking defects, including a partial defect in the sorting of carboxypeptidase Y (CPY) from the Golgi to the vacuole (12) and defects in the recycling of the exocytic vSNARE Snc1p and the lipophilic dye FM4-64 (14). These defects appear to be the result of an excessive level of activated Ypt1p, as overexpression of Ypt1p exaggerates each of the trafficking defects of a gyp1⌬ strain, leading to lethality.…”

“…Conversely, a partial loss of function mutation in ypt1 suppresses the growth and trafficking defects of a gyp1⌬ strain. Furthermore, the gyp1⌬ trafficking defects can be largely mimicked by a ypt1 allele deficient in GTP hydrolysis (12,14). The effects of the loss of Gyp1p on trafficking may relate to a role for Ypt1p and one of its effectors, the conserved oligomeric Golgi (COG) complex, in transport from the endosome to the Golgi (22).…”

“…Ypt1p is associated with the Golgi, where it regulates endoplasmic reticulum (ER) to Golgi and intra-Golgi traffic, as well as traffic from the endosome to the Golgi (10,11). Gyp1p is also associated with the Golgi, and phenotypic, genetic, and kinetic studies indicate that Gyp1p acts at that site to downregulate Ypt1p by stimulating its GTPase activity (12)(13)(14)(15). In contrast, Gyp1p shows no stimulation of the GTPase activity of Ypt32p (16,17).…”