Residue-by-residue analysis of cotranslational membrane protein integration<i>in vivo</i>

Nicolaus, Felix; Metola, Ane; Mermans, Daphne; Liljenström, Amanda; Krč, Ajda; Abdullahi, Salmo Mohammed; Zimmer, Matthew; Miller, Thomas F.; Heijne, Gunnar von

doi:10.1101/2020.09.27.315283

Cited by 7 publications

(27 citation statements)

References 36 publications

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“…3Ci) [64]. We speculate that hydrophobic signal-anchor sequences [66,67], some of which can also reorient inside the Sec61 translocon [68], will be subject to pulling forces comparable to those experienced by signal sequences during their membrane insertion at the ER [65,69]. Likewise, it seems plausible that the profiles and strength of the pulling forces experienced by signal sequences and/or signal anchors may be influenced by the drivers and determinants of signal orientation/ TMD topology, such as the 'positive-inside' rule, the degree of N-terminal folding and, perhaps, even the lipid composition of the bilayer [7,8].…”

Section: Gating Of the Sec61 Complexmentioning

confidence: 98%

“…By subjecting a broader range of TRAP-and Sec62/ 63-dependent clients of the Sec61 translocon [72,74] to detailed force-pulling studies [64,65,69,75,76], it should be possible to discover mechanistic detail about these gating assistants that is currently lacking. Currently unanswered questions include: is the constitutively Sec61-bound TRAP complex [78] the only 'gating assistant' capable of exerting a pulling force on RNCs (see [75]); can the Sec62/63 complex compensate for the loss of TRAP-mediated assistance; is TRAP-and/ or Sec62/63-assisted translocation used to regulate the flux of protein substrates through the Sec61 translocon [72,74]?…”

Section: Gating Of the Sec61 Complexmentioning

confidence: 99%

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Membrane protein biogenesis at the ER: the highways and byways

2021

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The Sec61 complex is the major protein translocation channel of the endoplasmic reticulum (ER), where it plays a central role in the biogenesis of membrane and secretory proteins. Whilst Sec61-mediated protein translocation is typically coupled to polypeptide synthesis, suggestive of significant complexity, an obvious characteristic of this core translocation machinery is its surprising simplicity. Over thirty years after its initial discovery, we now understand that the Sec61 complex is in fact the central piece of an elaborate jigsaw puzzle, which can be partly solved using new research findings. We propose that the Sec61 complex acts as a dynamic hub for cotranslational protein translocation at the ER, proactively recruiting a range of accessory complexes that enhance and regulate its function in response to different protein clients. It is now clear that the Sec61 complex does not have a monopoly on co-translational insertion, with some transmembrane proteins preferentially utilising the ER membrane complex instead. We also have a better understanding of post-insertion events, where at least one membrane-embedded chaperone complex can capture the newly inserted transmembrane domains of multi-span proteins and co-ordinate their assembly into a native structure. Having discovered this array of Sec61-associated components and competitors, our next challenge is to understand how they act together in order to expand the range and complexity of the membrane proteins that can be synthesised at the ER. Furthermore, this diversity of components and pathways may open up new opportunities for targeted therapeutic interventions designed to selectively modulate protein biogenesis at the ER. AbbreviationsBiP, immunoglobulin binding protein; CCDC47, coiled-coil domain containing 47 protein, also known as calumin; EMC, ER membrane complex; ER, endoplasmic reticulum; GET, guided entry of tail-anchored proteins; hSnd2, human SRP-independent protein 2, also known as TMEM208; NAC, nascent polypeptide-associated complex; NOMO, nodal modulating protein; PAT, protein associated with the ER translocon; RNC, ribosome-nascent chain; SGTA, small glutamine-rich tetratricopeptide repeat-containing protein alpha; SND, SRPindependent proteins or pathway; SPC, signal peptidase complex; SRP, signal recognition particle; TA, tail-anchored; TMCO1, transmembrane and coiled-coil domains 1 protein; TMD, transmembrane domain; TMEM147, transmembrane protein 147; TMEM208, transmembrane protein 208; TMP, transmembrane protein; TRAM, translocating chain-associated membrane protein; TRAP, transloconassociated protein; TRC40, transmembrane recognition complex of 40 kDa, also known as Asna1.

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Section: Gating Of the Sec61 Complexmentioning

confidence: 98%

Section: Gating Of the Sec61 Complexmentioning

confidence: 99%

Membrane protein biogenesis at the ER: the highways and byways

2021

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“…Peak II thus represents the membrane insertion of TMH2. N start values obtained with the SecM( Ms ) AP are typically ~5 residues larger than those obtained with the weaker SecM( Ec ) AP [33], and the two TMHs thus probably reach the SecYEG translocon when their N-termini are ~45 residues away from the PTC, as seen for other E. coli inner membrane proteins [46].…”

Section: Resultsmentioning

confidence: 99%

Cotranslational folding of a periplasmic protein domain inEscherichia coli

Sandhu

Hedman

Cymer

et al. 2021

Preprint

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In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA - a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide - to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB′s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45-50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.

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“…We focused on the analysis of cyclic homomers that do not localise to plasma or endomembrane systems, because of the competing hydrophobic forces exerted by protein-lipid interactions. Cotranslational membrane insertion of complexes is likely an active process requiring, for example, shielding factors 63,64 , rather than being solely driven by hydrophobic surface area. Despite the relatively limited number of structures available, we detect a significant difference in the mean subunit-to-subunit interface area among soluble members of the cyclic symmetry group (Figure 1B), with a 19% increase in the mean of cotranslationally forming members (p = 0.03, Wilcoxon rank-sum test).…”

Section: Cotranslationally Forming Homomers Are Characterised By Large Interfacesmentioning

confidence: 99%

Large protein complex interfaces have evolved to promote cotranslational assembly

Badonyi

Marsh

2021

Preprint

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Assembly pathways of protein complexes should be precise and efficient to minimise misfolding and unwanted interactions with other proteins in the cell. One way to achieve this is by seeding complex assembly during translation via nascent chain engagement. Here, we considered the possibility that the propensity of subunits to cotranslationally assemble is ingrained within the interface hierarchy of protein complexes. Using a combination of proteome-specific structure data and assembly-onset positions determined by ribosome profiling, we show that larger interfaces are prioritised in the course of cotranslational assembly. We observe that this effect is not exclusive to homomeric complexes, but appears to drive the assembly of heteromeric subunits, to the extent that interface size differences are detectable between N and C-terminal locations, with the former being larger on average. We provide explanations to this phenomenon and discuss its importance in a biological context.

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Residue-by-residue analysis of cotranslational membrane protein integrationin vivo

Cited by 7 publications

References 36 publications

Membrane protein biogenesis at the ER: the highways and byways

Membrane protein biogenesis at the ER: the highways and byways

Cotranslational folding of a periplasmic protein domain inEscherichia coli

Large protein complex interfaces have evolved to promote cotranslational assembly

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