Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein-lipid interactions are critical during translocon-mediated membrane insertion.

Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position-dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon-mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.

A substantial proportion of the genome encodes membrane proteins that are delivered to the endoplasmic reticulum by dedicated targeting pathways1. Membrane proteins that fail targeting must be rapidly degraded to avoid aggregation and disruption of cytosolic protein homeostasis2,3. The mechanisms of mislocalized protein (MLP) degradation are unknown. Here, we reconstitute MLP degradation in vitro to identify factors involved in this pathway. We find that nascent membrane proteins tethered to ribosomes are not substrates for ubiquitination unless they are released into the cytosol. Their inappropriate release results in capture by the Bag6 complex, a recently identified ribosome-associating chaperone4. Bag6 complex capture depends on unprocessed or non-inserted hydrophobic domains that distinguish MLPs from potential cytosolic proteins. A subset of these Bag6 clients is transferred to TRC40 for membrane insertion, while the remainder are rapidly ubiquitinated. Depletion of the Bag6 complex impairs efficient ubiquitination selectively of MLPs. Thus, by its presence on ribosomes synthesizing nascent membrane proteins, the Bag6 complex links targeting and ubiquitination pathways. We propose that such coupling permits fast-tracking of MLPs for degradation without futile engagement of cytosolic folding machinery.

An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson’s disease1. Recent studies of the Parkinson’s disease associated genes PINK1 (ref. 2) and parkin (PARK2, ref. 3) indicate that they may act in a quality control pathway preventing the accumulation of dysfunctional mitochondria4–8. Here we elucidate regulators that have an impact on parkin translocation to damaged mitochondria with genome-wide small interfering RNA (siRNA) screens coupled to high-content microscopy. Screening yielded gene candidates involved in diverse cellular processes that were subsequently validated in low-throughput assays. This led to characterization of TOMM7 as essential for stabilizing PINK1 on the outer mitochondrial membrane following mitochondrial damage. We also discovered that HSPA1L (HSP70 family member) and BAG4 have mutually opposing roles in the regulation of parkin translocation. The screens revealed that SIAH3, found to localize to mitochondria, inhibits PINK1 accumulation after mitochondrial insult, reducing parkin translocation. Overall, our screens provide a rich resource to understand mitochondrial quality control.