Contribution of Cotranslational Folding Defects to Membrane Protein Homeostasis

Roushar, Francis J.; Gruenhagen, Timothy C.; Penn, Wesley D.; Li, Bian; Meiler, Jens; Jastrzębska, Beata; Schlebach, Jonathan P.

doi:10.1021/jacs.8b08243

Cited by 27 publications

(69 citation statements)

References 70 publications

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“…S3A) (18). Mutations that promote the formation of these aberrant topomers have been found to decrease the surface expression of rhodopsin (18). A comparison of these putative topological states reveals that the eight C-terminal residues of TM7 (residues 302 to 309) are embedded within the membrane in the native topomer but solvated by water in these two non-native topological states ( fig.…”

Section: Effects Of Mutations On the Energetics Of Coand Posttranslatmentioning

confidence: 99%

“…Rhodopsin binds its retinal cofactor with an estimated K d (dissociation constant) of 25 pM (27), and the stabilization afforded by the thermodynamic coupling between binding and folding is known to enhance the cellular expression of rhodopsin (28). However, topological defects in TM7 prevent the formation of the native retinal binding pocket (18). As a result, the stabilization afforded by retinal binding cannot compensate for the proteostatic effects of mutations that disrupt the topology of TM7 (18).…”

Section: Asymmetric Influence Of Retinal On the Surface Expression Ofmentioning

confidence: 99%

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Probing biophysical sequence constraints within the transmembrane domains of rhodopsin by deep mutational scanning

et al. 2020

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Membrane proteins must balance the sequence constraints associated with folding and function against the hydrophobicity required for solvation within the bilayer. We recently found the expression and maturation of rhodopsin are limited by the hydrophobicity of its seventh transmembrane domain (TM7), which contains polar residues that are essential for function. On the basis of these observations, we hypothesized that rhodopsin’s expression should be less tolerant of mutations in TM7 relative to those within hydrophobic TM domains. To test this hypothesis, we used deep mutational scanning to compare the effects of 808 missense mutations on the plasma membrane expression of rhodopsin in HEK293T cells. Our results confirm that a higher proportion of mutations within TM7 (37%) decrease rhodopsin’s plasma membrane expression relative to those within a hydrophobic TM domain (TM2, 25%). These results in conjunction with an evolutionary analysis suggest solvation energetics likely restricts the evolutionary sequence space of polar TM domains.

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Section: Effects Of Mutations On the Energetics Of Coand Posttranslatmentioning

confidence: 99%

Section: Asymmetric Influence Of Retinal On the Surface Expression Ofmentioning

confidence: 99%

Probing biophysical sequence constraints within the transmembrane domains of rhodopsin by deep mutational scanning

et al. 2020

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“…TMDs of WRB that slowly flips into the ER lumen efficiently assemble with CAML. Previous studies 438 have shown that a charged residue in a TMD of membrane protein is often required for making an 439 ionic bond with an opposite charge in the partner TMD of membrane protein (Cosson et al, 1991;440 Feige and Hendershot, 2013; Roushar et al, 2019). Our data suggest that charged residues in TMDs 441 may also be evolved to interact with membrane protein holdases, thus allowing efficient assembly 442 with partner TMDs.…”

Section: Discussion 391mentioning

confidence: 75%

The C-terminal tail guides assembly and degradation of membrane proteins

Sun

Mariappan²

2019

Preprint

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SUMMARYA large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multi-protein complexes, but little is known about the mechanisms required for either assembly or degradation of unassembled membrane proteins. We find that C-terminal transmembrane domains (C-TMDs) with shorter tails are inefficiently inserted into the ER membrane since the translation is terminated before they emerge from ribosomes. These TMDs of insufficient hydrophobicity are post-translationally retained by the Sec61 translocon, thus providing a time window for efficient assembly with TMDs from partner membrane proteins. The unassembled C-TMDs are slowly flipped into the ER lumen. While the luminal chaperone BiP captures flipped C-TMDs with long tails and routes them to the ER-associated quality control, C-TMDs with shorter tails are diffused into the nuclear membrane. Thus, our studies suggest that C-terminal tails harbor important biochemical features for both biosynthesis and quality control of membrane protein complexes.

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“…Chimeric Lep proteins were generated by in vitro translation as was described previously. 45 Briefly, mRNA for each chimeric Lep protein was produced from plasmids using the RiboMAX RNA production system in accordance with the manufacturer's instructions (Promega, Madison, WI). Lep proteins were then produced from mRNA by in vitro translation using rabbit reticulocyte lysate (Promega, Madison, WI) supplemented with canine pancreatic rough microsomes (tRNA probes, College Station, TX) and EasyTag 35 S methionine (PerkinElmer, Waltham, MA).…”

Section: In Vitro Translation Of Chimeric Lep Proteinsmentioning

confidence: 99%

Cotranslational Folding Stimulates Programmed Ribosomal Frameshifting in the Alphavirus Structural Polyprotein

Harrington

Zimmer

Chamness

et al. 2019

Preprint

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Viruses maximize their genetic coding capacity through a variety of biochemical mechanisms including programmed ribosomal frameshifting (PRF), which facilitates the production of multiple proteins from a single transcript. PRF is typically stimulated by structural elements within the mRNA that generate mechanical tension between the transcript and ribosome. However, in this work we show that the forces generated by the cotranslational folding of the nascent polypeptide chain can also enhance PRF. Using an array of biochemical, cellular, and computational techniques, we first demonstrate that the Sindbis virus structural polyprotein forms two competing topological isomers during biosynthesis at the ribosome-translocon complex. We then show that the formation of one of these topological isomers is linked to PRF. Coarse-grained molecular dynamic simulations reveal that the translocon-mediated membrane integration of a transmembrane domain upstream from the ribosomal slip-site generates a force on the nascent polypeptide chain that scales with observed frameshifting. Together, our results demonstrate that cotranslational folding of this protein generates a tension that stimulates PRF. To our knowledge, this constitutes the first example in which the conformational state of the nascent chain has been linked to PRF. These findings raise the possibility that, in addition to RNA-mediated translational recoding, a variety of cotranslational folding and/ or binding events may also stimulate PRF.

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Contribution of Cotranslational Folding Defects to Membrane Protein Homeostasis

Cited by 27 publications

References 70 publications

Probing biophysical sequence constraints within the transmembrane domains of rhodopsin by deep mutational scanning

Probing biophysical sequence constraints within the transmembrane domains of rhodopsin by deep mutational scanning

The C-terminal tail guides assembly and degradation of membrane proteins

Cotranslational Folding Stimulates Programmed Ribosomal Frameshifting in the Alphavirus Structural Polyprotein

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