Cooperation of mitochondrial and ER factors in quality control of tail-anchored proteins.

Verena Dederer,Anna Gesine Huhn,Marius K Lemberg,Voytek Okreglak,Anton Khmelinskii,Michael Knop

doi:10.7554/elife.45506

Abstract

Tail-anchored (TA) proteins insert post-translationally into the endoplasmic reticulum (ER), the outer mitochondrial membrane (OMM) and peroxisomes. Whereas the GET pathway controls ER-targeting, no dedicated factors are known for OMM insertion, posing the question of how accuracy is achieved. The mitochondrial AAA-ATPase Msp1 removes mislocalized TA proteins from the OMM, but it is unclear, how Msp1 clients are targeted for degradation. Here we screened for factors involved in degradation of TA proteins mislocalized to mitochondria. We show that the ER-associated degradation (ERAD) E3 ubiquitin ligase Doa10 controls cytoplasmic level of Msp1 clients. Furthermore, we identified the uncharacterized OMM protein Fmp32 and the ectopically expressed subunit of the ER-mitochondria encounter structure (ERMES) complex Gem1 as native clients for Msp1 and Doa10. We propose that productive localization of TA proteins to the OMM is ensured by complex assembly, while orphan subunits are extracted by Msp1 and eventually degraded by Doa10.

Highlights

Correct localization of proteins is essential to ensure their functionality and to establish the identity of individual cellular organelles
To be able to measure Pex15D30 turnover in high-throughput in different mutants, we fused Pex15D30 to a tandem fluorescent protein timer consisting of the fast-maturing superfolder GFP and the slower-maturing mCherry (Figure 1A) (Khmelinskii et al, 2012)
Deletion of GET3, which was previously linked to trafficking of full-length Pex15 and shows a negative genetic interaction with msp1D (Costanzo et al, 2010; Okreglak and Walter, 2014), caused a significant increase in superfolder GFP (sfGFP) level and mCherry/sfGFP ratio. tandem fluorescent protein timer (tFT)-Pex15D30 was significantly stabilized in the absence of the ER-resident P-type ATPase Spf1, the pheromone regulated membrane protein Prm1 and, interestingly, the ER-resident E3 ubiquitin ligase Doa10, its associated E2 ubiquitin-conjugating enzyme Ubc7 and its tethering factor Cue1 (Figure 1B)

Summary

Introduction

Correct localization of proteins is essential to ensure their functionality and to establish the identity of individual cellular organelles. Membrane proteins that are not recognized by SRP are captured by other cytosolic chaperons, which keep these proteins in an unfolded state guiding them to the ER, mitochondria or peroxisomes post-translationally (Aviram and Schuldiner, 2017; Hegde and Keenan, 2011; Wasilewski et al, 2017). Tail-anchored (TA) proteins are a specific class of membrane proteins that have a single transmembrane (TM) domain at their very C-terminus. They are involved in various cellular processes such as membrane fusion, protein translocation and regulation of apoptosis (Antonsson, 2001; Beilharz et al, 2003; Burri and Lithgow, 2004; Chen and Scheller, 2001; Kalbfleisch et al, 2007). A cytosolic pre-targeting complex comprising Sgt, Get, and Get captures the TM segment of a TA protein after it emerges from the ribosome and loads it onto Get (Mariappan et al, 2010; Mateja et al, 2009; Schuldiner et al, 2008)

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Conclusion