Msp1 Protects Organelles Through Quality Control of Excess Membrane Proteins

Abstract

Quality control (QC) of membrane proteins is a critical but ill-understood process where undesirable proteins are sequestered or eliminated to protect cellular homeostasis. A recently described membrane protein QC pathway centers around Msp1, an ATPase Associated with diverse cellular Activities (AAA) family protein which resides at mitochondria and peroxisomes. Msp1 protects mitochondria from accumulating “mislocalized” tail-anchored (TA) proteins which normally reside in the endoplasmic reticulum (ER) or peroxisomes, but occasionally mis-target to mitochondria. We asked how Msp1 identifies its mislocalized substrates while avoiding turnover of the higher-abundance pool of similar but normally mitochondrial TA proteins. We used a combination of quantitative microscopy and molecular techniques to examine how Msp1 identifies its substrates. First, we found that Msp1 can eliminate the normally peroxisomal TA protein Pex15 after it integrates into the lipid bilayer at mitochondria. Next, we observed that Msp1 can eliminate peroxisomal Pex15 if either Msp1 or Pex15 is overexpressed. We found that Pex15 binds to Pex3 at peroxisomes and is thus shielded from Msp1, but excess Pex15 is unbound and therefore susceptible to turnover by Msp1. Using mutagenesis, we determined that the Pex15 TMS but not the Msp1 TMS sequence is required for Msp1 to engage Pex15. Instead, genetic screening revealed conserved residues between Msp1’s TMS and its AAA domain required for its activity and perhaps for substrate engagement. Interestingly, Msp1 levels are connected to accurate substrate engagement: reducing Msp1 expression reduces substrate turnover and disrupts peroxisomal function, whereas increasing Msp1 levels interferes with peroxisomal physiology through spurious recognition of nascent functional Pex15. Our data therefore reveal a concentration-based scheme by which Msp1 selectively identifies excess or mis-targeted membrane proteins which are unable to engage their binding partners.