Systematic Profiling of Temperature- and Retinal-Sensitive Rhodopsin Variants by Deep Mutational Scanning

Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of temperature-sensitive variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and an investigational corrector (9-cis-retinal) on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures is correlated with the response to retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants within this helix appear to disrupt a network of hydrogen bonds that stabilizes a native helical kink. By comparison, mutants that alter a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest this insensitivity primarily arises from an abundance of mutations that enhance its membrane integration, stabilize its native conformation, and/ or perturb the retinal binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings elucidate various factors that mediate the sensitivity of genetic variants to temperature and to small molecule correctors.

Differential modes of orphan subunit recognition for the WRB/CAML complex

A large proportion of membrane proteins must be assembled into oligomeric complexes for function. How this process occurs is poorly understood, but it is clear that complex assembly must be tightly regulated to avoid accumulation of orphan subunits with potential cytotoxic effects. We interrogated assembly in mammalian cells using a model system of the WRB/CAML complex: an essential insertase for tail-anchored proteins in the endoplasmic reticulum (ER). Our data suggests that the stability of each subunit is differentially regulated. In WRB’s absence, CAML folds incorrectly, causing aberrant exposure of a hydrophobic transmembrane domain to the cytosol. When present, WRB can post-translationally correct the topology of CAML both in vitro and in cells. In contrast, WRB can independently fold correctly, but is still degraded in the absence of CAML. We therefore propose at least two distinct regulatory pathways for the surveillance of orphan subunits during complex assembly in the mammalian ER.

The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved

ABSTRACTTransmembrane domains (TMDs) from single-spanning membrane proteins are commonly viewed as membrane anchors for functional domains. Influenza virus neuraminidase (NA) exemplifies this concept, as it retains enzymatic function upon proteolytic release from the membrane. However, the subtype 1 NA TMDs have become increasingly more polar in human strains since 1918, which suggests that selection pressure exists on this domain. Here, we investigated the N1 TMD-head domain relationship by exchanging a prototypical “old” TMD (1933) with a “recent” (2009), more polar TMD and an engineered hydrophobic TMD. Each exchange altered the TMD association, decreased the NA folding efficiency, and significantly reduced viral budding and replication at 37°C compared to at 33°C, at which NA folds more efficiently. Passaging the chimera viruses at 37°C restored the NA folding efficiency, viral budding, and infectivity by selecting for NA TMD mutations that correspond with their polar or hydrophobic assembly properties. These results demonstrate that single-spanning membrane protein TMDs can influence distal domain folding, as well as membrane-related processes, and suggest the NA TMD in H1N1 viruses has become more polar to maintain compatibility with the evolving enzymatic head domain.IMPORTANCEThe neuraminidase (NA) protein from influenza A viruses (IAVs) functions to promote viral release and is one of the major surface antigens. The receptor-destroying activity in NA resides in the distal head domain that is linked to the viral membrane by an N-terminal hydrophobic transmembrane domain (TMD). Over the last century, the subtype 1 NA TMDs (N1) in human H1N1 viruses have become increasingly more polar, and the head domains have changed to alter their antigenicity. Here, we provide the first evidence that an “old” N1 head domain from 1933 is incompatible with a “recent” (2009), more polar N1 TMD sequence and that, during viral replication, the head domain drives the selection of TMD mutations. These mutations modify the intrinsic TMD assembly to restore the head domain folding compatibility and the resultant budding deficiency. This likely explains why the N1 TMDs have become more polar and suggests the N1 TMD and head domain have coevolved.

Synthetic Peptides as Structural Maquettes of Angiotensin-I Converting Enzyme Catalytic Sites

The rational design of synthetic peptides is proposed as an efficient strategy for the structural investigation of crucial protein domains difficult to be produced. Only after half a century since the function of ACE was first reported, was its crystal structure solved. The main obstacle to be overcome for the determination of the high resolution structure was the crystallization of the highly hydrophobic transmembrane domain. Following our previous work, synthetic peptides and Zinc(II) metal ions are used to build structural maquettes of the two Zn-catalytic active sites of the ACE somatic isoform. Structural investigations of the synthetic peptides, representing the two different somatic isoform active sites, through circular dichroism and NMR experiments are reported.

Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface

Angiotensin I-converting enzyme (ACE; CD143, EC 3.4.15.1) is a type-1 integral membrane protein that can also be released into extracellular fluids (such as plasma, and seminal and cerebrospinal fluids) as a soluble enzyme following cleavage mediated by an unidentified protease(s), referred to as ACE secretase, in a process known as ‘shedding'. The effects of monoclonal antibodies (mAbs) to eight different epitopes on the N-terminal domain of ACE on shedding was investigated using Chinese hamster ovary cells (CHO cells) expressing an ACE transgene and using human umbilical vein endothelial cells. Antibody-induced shedding of ACE was strongly epitope-specific: most of the antibodies increased the shedding by 20–40%, mAbs 9B9 and 3A5 increased the shedding by 270 and 410% respectively, whereas binding of mAb 3G8 decreased ACE shedding by 36%. The ACE released following mAb treatment lacked a hydrophobic transmembrane domain anchor. The antibody-induced shedding was completely inhibited at 4°C and by zinc chelation using 1,10-phenanthroline, suggesting involvement of a metalloprotease in this process. A hydroxamate-based metalloprotease inhibitor (batimastat, BB-94) was 15 times more efficacious in inhibiting mAb-induced ACE shedding than basal (constitutive) ACE release. Treatment of CHO-ACE cells with BB-94 more effectively prevented elevation in antibody-dependent (but not basal) ACE release induced by 3,4-dichloroisocoumarin and iodoacetamide. These data suggest that different secretases might be responsible for ACE release under basal compared with antibody-induced shedding. Further experiments with more than 40 protease inhibitors suggest that calpains, furin and the proteasome may participate in this process.

Analysis of Structural Signals Conferring Localisation of Pig OST48 to the Endoplasmic Reticulum

Pig liver oligosaccharyltransferase (OST) is a heterooligomeric protein complex responsible for the cotranslational transfer of GlcNAc[2]Man[9]Glc[3] from Dol PP onto specific asparagine residues in the nascent polypeptide. OST48, one of the catalytic subunits in this complex, exerts a typical type I membrane topology, containing a large luminal domain, a hydrophobic transmembrane domain and a short cytosolic peptide tail. Because OST48 is found within the endoplasmic reticulum (ER) when overexpressed in COS-1 cells, we carried out experiments to identify structural signals potentially capable of directing ERtargeting, using OST48 mutants and hybrid proteins consisting of individual OST48 domains and Man[9] mannosidase. Immunofluorescence microscopy showed that OST48 mutants in which the Cterminal lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48?K) could be detected on the cell surface. This indicates that these two lysine residues are sufficient for conferring ERresidency on OST48. The doublelysine motif operates only when exposed cytosolically, where it acts as a relocation signal rather than causing retention. OST48?K-3, when coexpressed in COS-1 cells together with myctagged ribophorin I, was quantitatively retained in the ER. By contrast, coexpression in the presence of ribophorin I resulted in no reduction of cell surface fluorescence for the OMO?K-5 chimera containing the cytosolic and transmembrane domain of OST48 attached to the Cterminus of the Man[9]mannosidase luminal domain. Thus ERlocalisation of OST48 is probably brought about by complex formation with ribophorin I and this most likely involves the luminal domains of both proteins. Consequently, the doublelysine motif in the cytosolic domain of OST48 is unlikely to have a primary function except being involved in recapture of molecules which have escaped from the ER.