Regulation of the Pathway for Guided Entry of Tail-anchored Proteins

Abstract

Tail-anchored (TA) proteins possess numerous essential functions throughout the secretory and endocytic pathways, where they are anchored to membranes by a single hydrophobic transmembrane domain (TMD) at their C-terminus. The biogenesis of TA proteins occurs post-translationally via the GET (Guided Entry of Tail-anchored proteins) pathway, which promotes efficient, ATP hydrolysis-dependent, cycles of TA protein targeting and insertion at the endoplasmic reticulum (ER). However, it is unclear how GET pathway components react to conditions that might compromise the integration of TA proteins at the ER membrane and whether such a response affects the fate of the precursors. This study investigates the possibility that shifts in the intracellular localisation of the soluble TA protein delivery factor Get3 reflect a mechanism by which the GET pathway is regulated. Live cell imaging revealed that Get3 localises to cytosolic foci that contain components of the cytosolic Get4-Get5-Sgt2 complex and molecular chaperones upon glucose depletion. This process was found to be reversible, dependent on Get4/Get5 and influenced by the ability of Get3 to hydrolyse ATP. When integration at the ER membrane was blocked, glucose depletion also triggered the accumulation of mislocalised TA proteins at these sites. Quantitative analysis of TA protein levels revealed that upon glucose re-addition after a period of withdrawal, a proportion of TA proteins are eliminated in a Get3- and Get4/Get5-dependent manner. These data suggest that during glucose depletion, Get3 moves to deposition sites for aggregation-prone proteins, where it may maintain mislocalised TA proteins in a state that is amenable to subsequent processing. Furthermore, perturbations of deubiquitination were found to interfere with the localisation behaviour of Get3, and the elimination of mislocalised TA proteins, suggesting that the GET pathway and ubiquitin-mediated degradation are closely associated. Taken together with existing work in the field, these data support a model in which Get3 has a dual function that is regulated by cellular energy levels, and implicate the cytosolic GET complex in the quality control of TA proteins during their biogenesis.

Date of Award	1 Aug 2013
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Stephen High (Supervisor) & Blanche Schwappach (Supervisor)