Contribution of Cotranslational Folding Defects to Membrane Protein Homeostasis

Abstract

Membrane proteins are prone to misfolding and degradation within the cell, yet the nature of the conformational defects involved in this process remain poorly understood. The earliest stages of membrane protein folding are mediated by the Sec61 translocon, a molecular machine that facilitates the lateral partitioning of the polypeptide into the membrane. Proper membrane integration is an essential prerequisite for folding of the nascent chain. However, the marginal energetic drivers of this reaction suggest the translocon may operate with modest fidelity. In this work, we employed biophysical modeling in conjunction with quantitative biochemical measurements in order to evaluate the extent to which cotranslational folding defects influence membrane protein homeostasis. Protein engineering was employed to selectively perturb the topological energetics of human rhodopsin, and the expression and cellular trafficking of engineered variants were quantitatively compared. Our results reveal clear relationships between topological energetics and the efficiency of rhodopsin biogenesis, which appears to be limited by the propensity of a polar transmembrane domain to achieve its correct topological orientation. Though the polarity of this segment is functionally constrained, we find that its topology can be stabilized in a manner that enhances biogenesis without compromising the functional properties of rhodopsin. Furthermore, sequence alignments reveal this topological instability has been conserved throughout the course of evolution. These results suggest that topological defects significantly contribute to the inefficiency of membrane protein folding in the cell. Additionally, our findings suggest that the marginal stability of rhodopsin may represent an evolved trait.

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

“…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.…”

“…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).…”

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

“…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.…”

Cotranslational Folding Stimulates Programmed Ribosomal Frameshifting in the Alphavirus Structural Polyprotein

“…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).…”