Targeting of the membrane‐anchored Rab GAP (GTPase accelerating protein) Gyp8 to peroxisomes is regulated by the AAA ATPase Msp1

Abstract

Eukaryotic cells use vesicular transport to build and maintain membrane‐bound organelles housing specific biochemical functions. Rab GTPase signaling proteins (Rabs) are key regulators of vesicular transport, controlling where and when membranes dock and fuse, ensuring lipid and protein cargoes are delivered to appropriate destinations. Rabs depend upon GTPase accelerating proteins (GAPs) to trigger GTP hydrolysis and return the signaling active, GTP‐bound Rab to its inactive, GDP‐bound state. Rab GAPs contribute to the efficiency and fidelity of transport pathways, and defective Rab GAPs are implicated in a variety of human diseases. Rab GAPs tend to be cytosolic proteins that transiently localize at membranes to survey for their client Rab. We report that the yeast Rab GAP Gyp8 is an atypical transmembrane (TM) GAP whose association with peroxisomes is regulated by the AAA ATPase Msp1, a chaperone that functions to remove tail‐anchored proteins from peroxisomes and mitochondria. Computational analysis of Gyp8 predicted a single‐pass TM domain near the carboxy terminus, characteristic of a tail‐anchored protein. Fluoresence microscopy indicated that GFP‐tagged Gyp8 co‐localized with endoplasmic reticulum (ER) and peroxisomal markers in wild type cells. In the absence of peroxisomes, GFP‐Gyp8 redistributed to the ER. Loss of Msp1 chaperone function resulted in mislocalization of GFP‐Gyp8 to mitochondrial membranes. Subcellular fractionation biochemically demonstrated that Gyp8 localized exclusively to a membrane fraction containing peroxisomes. Truncation analyses of Gyp8 indicated that the TM and luminal domains are necessary and sufficient to direct localization to peroxisomes. Growth and viability experiments of cells lacking specific Rab GAPs indicate a peroxisome‐specific carbon utilization defect in cells lacking Gyp8. These data represent the first report in any experimental system of a Rab GAP that localizes to peroxisomes.Support or Funding InformationFunded by grants from the CSUSB Office of Student Research and Associated Students Inc.