Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology.

Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse proteins remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse ⍺-helical substrates reveals that these components are organized into distinct targeting pathways that act on substrates based on their topology. NAC is required for the efficient targeting of polytopic proteins, whereas signal-anchored proteins require TTC1, a cytosolic chaperone that physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, the targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems.

Mechanism of signal-anchor triage during early steps of membrane protein insertion.

Most membrane proteins use their first transmembrane domain, known as a signal anchor (SA), for co-translational targeting to the endoplasmic reticulum (ER) via the signal recognition particle (SRP). The SA then inserts into the membrane using either the Sec61 translocation channel or the ER membrane protein complex (EMC) insertase. How EMC and Sec61 collaborate to ensure SA insertion in the correct topology is not understood. Using site-specific crosslinking, we detect a pre-insertion SA intermediate adjacent to EMC. This intermediate forms after SA release from SRP but before ribosome transfer to Sec61. The polypeptide's N-terminal tail samples a cytosolic vestibule bordered by EMC3, from where it can translocate across the membrane concomitant with SA insertion. The ribosome then docks on Sec61, which has an opportunity to insert those SAs skipped by EMC. These results suggest that EMC acts between SRP and Sec61 to triage SAs for insertion during membrane protein biogenesis.

Function of the Omp85 Superfamily of Outer Membrane Protein Assembly Factors and Polypeptide Transporters.

The Omp85 protein superfamily is found in the outer membrane (OM) of all gram-negative bacteria and eukaryotic organelles of bacterial origin. Members of the family catalyze both the membrane insertion of ß-barrel proteins and the translocation of proteins across the OM. Although the mechanism(s) by which these proteins function is unclear, striking new insights have emerged from recent biochemical and structural studies. In this review we discuss the entire Omp85 superfamily but focus on the function of the best-studied member, BamA, which is an essential and highly conserved component of the bacterial barrel assembly machinery (BAM). Because BamA has multiple functions that overlap with those of other Omp85 proteins, it is likely the prototypical member of the Omp85 superfamily. Furthermore, BamA has become a protein of great interest because of the recent discovery of small-molecule inhibitors that potentially represent an important new class of antibiotics.

Oxa1 Superfamily: New Members Found in the ER.

Oxa1/Alb3/YidC family members promote the insertion of proteins into the mitochondrial inner membrane, the chloroplast thylakoid membrane, and the bacterial plasma membrane. Remarkably, two recent studies identify new Oxa1 homologs that reside in the endoplasmic reticulum (ER) and function in ER membrane protein biogenesis.

A Limitation of Using Dithionite Quenching to Determine the Topology of Membrane-inserted Proteins.

Determining the topology of membrane-inserted proteins and peptides often relies upon indirect fluorescent measurements. One such technique uses NBD, an environmentally sensitive fluorophore that can be covalently linked to proteins. Relative to a hydrophilic environment, NBD in a hydrophobic environment shows an increase in emission intensity and a shift to shorter wavelengths. To gain further insight, NBD fluorescence can be chemically quenched using dithionite. As dithionite is an anion, it is only expected to penetrate the outer leaflet interfacial region and should be excluded from the hydrocarbon core, the inner leaflet, and the lumen of LUV. This assumption holds at neutral pH, where a large number of NBD/dithionite experiments are carried out. Here, we report control experiments in which LUV were directly labeled with NBD-PE to assess dithionite quenching in acidic conditions. Results showed that at acidic pH, dithionite moved more freely across the bilayer to quench the inner leaflet. For the buffer conditions used, dithionite exhibited a sharp change in behavior between pH 5.5 and 6.0. Therefore, in acidic conditions, dithionite could not differentiate in which leaflet the NBD resided.

C-terminal tail length guides insertion and assembly of membrane proteins.

A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multiprotein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ∼60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ∼80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins.

Partially inserted nascent chain unzips the lateral gate of the Sec translocon.

The Sec translocon provides the lipid bilayer entry for ribosome-bound nascent chains and thus facilitates membrane protein biogenesis. Despite the appreciated role of the native environment in the translocon:ribosome assembly, structural information on the complex in the lipid membrane is scarce. Here, we present a cryo-electron microscopy-based structure of bacterial translocon SecYEG in lipid nanodiscs and elucidate an early intermediate state upon insertion of the FtsQ anchor domain. Insertion of the short nascent chain causes initial displacements within the lateral gate of the translocon, where α-helices 2b, 7, and 8 tilt within the membrane core to "unzip" the gate at the cytoplasmic side. Molecular dynamics simulations demonstrate that the conformational change is reversed in the absence of the ribosome, and suggest that the accessory α-helices of SecE subunit modulate the lateral gate conformation. Site-specific cross-linking validates that the FtsQ nascent chain passes the lateral gate upon insertion. The structure and the biochemical data suggest that the partially inserted nascent chain remains highly flexible until it acquires the transmembrane topology.

C-terminal kink formation is required for lateral gating in BamA.

In Gram-negative bacteria, the outer membrane contains primarily ß-barrel transmembrane proteins and lipoproteins. The insertion and assembly of ß-barrel outer-membrane proteins (OMPs) is mediated by the ß-barrel assembly machinery (BAM) complex, the core component of which is the 16-stranded transmembrane ß-barrel BamA. Recent studies have indicated a possible role played by the seam between the first and last ß-barrel strands of BamA in the OMP insertion process through lateral gating and a destabilized membrane region. In this study, we have determined the stability and dynamics of the lateral gate through over 12.5 µs of equilibrium simulations and 4 µs of free-energy calculations. From the equilibrium simulations, we have identified a persistent kink in the C-terminal strand and observed spontaneous lateral-gate separation in a mimic of the native bacterial outer membrane. Free-energy calculations of lateral gate opening revealed a significantly lower barrier to opening in the C-terminal kinked conformation; mutagenesis experiments confirm the relevance of C-terminal kinking to BamA structure and function.

Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER.

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co- or post-translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients.

Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER.

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.

BamA ß16C strand and periplasmic turns are critical for outer membrane protein insertion and assembly.

Outer membrane (OM) ß-barrel proteins play important roles in importing nutrients, exporting wastes and conducting signals in Gram-negative bacteria, mitochondria and chloroplasts. The outer membrane proteins (OMPs) are inserted and assembled into the OM by OMP85 family proteins. In Escherichia coli, the ß-barrel assembly machinery (BAM) contains four lipoproteins such as BamB, BamC, BamD and BamE, and one OMP BamA, forming a 'top hat'-like structure. Structural and functional studies of the E. coli BAM machinery have revealed that the rotation of periplasmic ring may trigger the barrel ß1C-ß6C scissor-like movement that promote the unfolded OMP insertion without using ATP. Here, we report the BamA C-terminal barrel structure of Salmonella enterica Typhimurium str. LT2 and functional assays, which reveal that the BamA's C-terminal residue Trp, the ß16C strand of the barrel and the periplasmic turns are critical for the functionality of BamA. These findings indicate that the unique ß16C strand and the periplasmic turns of BamA are important for the outer membrane insertion and assembly. The periplasmic turns might mediate the rotation of the periplasmic ring to the scissor-like movement of BamA ß1C-ß6C, triggering the OMP insertion. These results are important for understanding the OMP insertion in Gram-negative bacteria, as well as in mitochondria and chloroplasts.

Hydrophilic microenvironment required for the channel-independent insertase function of YidC protein.

The recently solved crystal structure of YidC protein suggests that it mediates membrane protein insertion by means of an intramembrane cavity rather than a transmembrane (TM) pore. This concept of protein translocation prompted us to characterize the native, membrane-integrated state of YidC with respect to the hydropathic nature of its TM region. Here, we show that the cavity-forming region of the stage III sporulation protein J (SpoIIIJ), a YidC homolog, is indeed open to the aqueous milieu of the Bacillus subtilis cells and that the overall hydrophilicity of the cavity, along with the presence of an Arg residue on several alternative sites of the cavity surface, is functionally important. We propose that YidC functions as a proteinaceous amphiphile that interacts with newly synthesized membrane proteins and reduces energetic costs of their membrane traversal.

Pull-and-Paste of Single Transmembrane Proteins.

How complex cytoplasmic membrane proteins insert and fold into cellular membranes is not fully understood. One problem is the lack of suitable approaches that allow investigating the process by which polypeptides insert and fold into membranes. Here, we introduce a method to mechanically unfold and extract a single polytopic α-helical membrane protein, the lactose permease (LacY), from a phospholipid membrane, transport the fully unfolded polypeptide to another membrane and insert and refold the polypeptide into the native structure. Insertion and refolding of LacY is facilitated by the transmembrane chaperone/insertase YidC in the absence of the SecYEG translocon. Insertion into the membrane occurs in a stepwise, stochastic manner employing multiple coexisting pathways to complete the folding process. We anticipate that our approach will provide new means of studying the insertion and folding of membrane proteins and to mechanically reconstitute membrane proteins at high spatial precision and stoichiometric control, thus allowing the functional programming of synthetic and biological membranes.

Membrane insertion of F0 c subunit of F0F1 ATPase depends on glycolipozyme MPIase and is stimulated by YidC.

The F0 c subunit of F0F1 ATPase (F0-c) possesses two membrane-spanning stretches with N- and C-termini exposed to the periplasmic (extracellular) side of the cytoplasmic membrane of E. coli. Although F0-c insertion has been extensively analyzed in vitro by means of protease protection assaying, it is unclear whether such assays allow elucidation of the insertion process faithfully, since the membrane-protected fragment, an index of membrane insertion, is a full-length polypeptide of F0-c, which is the same as the protease-resistant conformation without membrane insertion. We found that the protease-resistant conformation could be discriminated from membrane-insertion by including octyl glucoside on protease digestion. By means of this system, we found that F0-c insertion depends on MPIase, a glycolipozyme involved in membrane insertion, and is stimulated by YidC. In addition, we found that acidic phospholipids PG and CL transform F0-c into a protease-resistant form, while MPIase prevents the acquisition of such a protease-resistant conformation.

YidC/Alb3/Oxa1 Family of Insertases.

The YidC/Alb3/Oxa1 family functions in the insertion and folding of proteins in the bacterial cytoplasmic membrane, the chloroplast thylakoid membrane, and the mitochondrial inner membrane. All members share a conserved region composed of five transmembrane regions. These proteins mediate membrane insertion of an assorted group of proteins, ranging from respiratory subunits in the mitochondria and light-harvesting chlorophyll-binding proteins in chloroplasts to ATP synthase subunits in bacteria. This review discusses the YidC/Alb3/Oxa1 protein family as well as their function in membrane insertion and two new structures of the bacterial YidC, which suggest a mechanism for membrane insertion by this family of insertases.

Use of Synchrotron Radiation Circular Dichroism to Analyze the Interaction and Insertion of Proteins into Bacterial Outer Membrane Vesicles.

Circular dichroism (CD) is a spectroscopic technique commonly used for the analysis of proteins. Particularly, it allows the determination of protein secondary structure content in various media, including the membrane environment. In this chapter, we present how CD applications can be used to analyze the interaction of proteins with bacterial outer membrane vesicles (OMVs). Most CD studies characterizing the structure of proteins inserted into membranes rely on artificial lipid bilayers, mimicking natural membranes. Nevertheless, these artificial models lack the important features of the true membrane, especially for the outer membrane of Gram-negative bacteria. These features include lipid diversity, glycosylation, and asymmetry. Here, we show how to analyze the interactions of proteins, either integral or peripheral, with OMVs in solution and with supported membranes of OMVs, using conventional CD and orientated circular dichroism (OCD). We explain how to decipher the spectroscopic signals to obtain information on the molecular structure of the protein upon its interaction with an OMV and through its potential insertion into an OMV membrane.

Structural analysis of the dynamic ribosome-translocon complex.

The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane proteins. Here, we leveraged recent advances in cryo-electron microscopy (cryo-EM) and structure prediction to derive insights into several novel configurations of the ribosome-translocon complex. We show how a transmembrane domain (TMD) in a looped configuration passes through the Sec61 lateral gate during membrane insertion; how a nascent chain can bind and constrain the conformation of ribosomal protein uL22; and how the translocon-associated protein (TRAP) complex can adjust its position during different stages of protein biogenesis. Most unexpectedly, we find that a large proportion of translocon complexes contains RAMP4 intercalated into Sec61's lateral gate, widening Sec61's central pore and contributing to its hydrophilic interior. These structures lead to mechanistic hypotheses for translocon function and highlight a remarkably plastic machinery whose conformations and composition adjust dynamically to its diverse range of substrates.

YajC, a predicted membrane protein, promotes Enterococcus faecium biofilm formation in vitro and in a rat endocarditis model.

Biofilm formation is a critical step in the pathogenesis of difficult-to-treat Gram-positive bacterial infections. We identified that YajC, a conserved membrane protein in bacteria, plays a role in biofilm formation of the clinically relevant Enterococcus faecium strain E1162. Deletion of yajC conferred significantly impaired biofilm formation in vitro and was attenuated in a rat endocarditis model. Mass spectrometry analysis of supernatants of washed ΔyajC cells revealed increased amounts in cytoplasmic and cell-surface-located proteins, including biofilm-associated proteins, suggesting that proteins on the surface of the yajC mutant are only loosely attached. In Streptococcus mutans YajC has been identified in complex with proteins of two cotranslational membrane protein-insertion pathways; the signal recognition particle (SRP)-SecYEG-YajC-YidC1 and the SRP-YajC-YidC2 pathway, but its function is unknown. In S. mutans mutation of yidC1 and yidC2 resulted in impaired protein insertion in the cell membrane and secretion in the supernatant. The E. faecium genome contains all homologous genes encoding for the cotranslational membrane protein-insertion pathways. By combining the studies in S. mutans and E. faecium, we propose that YajC is involved in the stabilization of the SRP-SecYEG-YajC-YidC1 and SRP-YajC-Yid2 pathway or plays a role in retaining proteins for proper docking to the YidC insertases for translocation in and over the membrane.

The Get1/2 insertase forms a channel to mediate the insertion of tail-anchored proteins into the ER.

Tail-anchored (TA) proteins contain a single C-terminal transmembrane domain (TMD) that is captured by the cytosolic Get3 in yeast (TRC40 in humans). Get3 delivers TA proteins to the Get1/2 complex for insertion into the endoplasmic reticulum (ER) membrane. How Get1/2 mediates insertion of TMDs of TA proteins into the membrane is poorly understood. Using bulk fluorescence and microfluidics assays, we show that Get1/2 forms an aqueous channel in reconstituted bilayers. We estimate the channel diameter to be ∼2.5 nm wide, corresponding to the circumference of two Get1/2 complexes. We find that the Get3 binding can seal the Get1/2 channel, which dynamically opens and closes. Our mutation analysis further shows that the Get1/2 channel activity is required to release TA proteins from Get3 for insertion into the membrane. Hence, we propose that the Get1/2 channel functions as an insertase for insertion of TMDs and as a translocase for translocation of C-terminal hydrophilic segments.

The insertase YidC chaperones the polytopic membrane protein MelB inserting and folding simultaneously from both termini.

The insertion and folding of proteins into membranes is crucial for cell viability. Yet, the detailed contributions of insertases remain elusive. Here, we monitor how the insertase YidC guides the folding of the polytopic melibiose permease MelB into membranes. In vivo experiments using conditionally depleted E. coli strains show that MelB can insert in the absence of SecYEG if YidC resides in the cytoplasmic membrane. In vitro single-molecule force spectroscopy reveals that the MelB substrate itself forms two folding cores from which structural segments insert stepwise into the membrane. However, misfolding dominates, particularly in structural regions that interface the pseudo-symmetric α-helical domains of MelB. Here, YidC takes an important role in accelerating and chaperoning the stepwise insertion and folding process of both MelB folding cores. Our findings reveal a great flexibility of the chaperoning and insertase activity of YidC in the multifaceted folding processes of complex polytopic membrane proteins.