Mapping out forces that act on transmembrane helices during membrane insertion

Despite the ubiquity of helical membrane proteins in nature and their pharmacological importance, the mechanisms guiding their folding remain unclear. We performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2–3 residues throughout the 178-residue transmembrane domain) of GlpG, a membrane-embedded rhomboid protease from Escherichia coli. The only clustering of significantly positive ϕ-values occurs at the cytosolic termini of transmembrane helices 1 and 2, which we identify as a compact nucleus. The three loops flanking these helices show a preponderance of negative ϕ-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3–6 yielded predominantly ϕ-values near zero, indicating that this part of the protein has denatured-state–level structure in the transition state. We propose that loops 1–3 undergo conformational rearrangements to position the folding nucleus correctly, which then drives folding of the rest of the domain. A compact N-terminal nucleus is consistent with the vectorial nature of cotranslational membrane insertion found in vivo. The origin of the interactions in the transition state that lead to a large number of negative ϕ-values remains to be elucidated.

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Do protein–lipid interactions determine the recognition of transmembrane helices at the ER translocon?

Biochemical Society Transactions ◽

10.1042/bst0331012 ◽

2005 ◽

Vol 33 (5) ◽

pp. 1012-1015 ◽

Cited By ~ 11

Author(s):

S.H. White ◽

G. von Heijne

Keyword(s):

Quantitative Information ◽

Membrane Insertion ◽

Transmembrane Helices ◽

Transmembrane Segment ◽

Hydrophobicity Scale ◽

Model Protein ◽

Target Membrane ◽

Correct Target ◽

Molecular Code

Membrane-protein integration, folding and assembly processes in vivo depend on complex targeting, translocation, chaperoning, and sorting machineries that somehow read the ‘molecular code’ built into the nascent polypeptide, ultimately producing a properly folded protein integrated into the correct target membrane. Although the main molecular constituents and the basic mechanistic principles of many of these machines are known in outline, the codes remain poorly defined and there is little quantitative information on how protein sequence affects the final structure of membrane proteins. By carefully designing model protein constructs, we have derived the first true biological hydrophobicity scale and have been able to get a first impression of how the position of a given type of residue within a transmembrane segment affects its ability to promote membrane insertion.

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Transmembrane helix hydrophobicity is an energetic barrier during the retrotranslocation of integral membrane ERAD substrates

Molecular Biology of the Cell ◽

10.1091/mbc.e17-03-0184 ◽

2017 ◽

Vol 28 (15) ◽

pp. 2076-2090 ◽

Cited By ~ 11

Author(s):

Christopher J. Guerriero ◽

Karl-Richard Reutter ◽

Andrew A. Augustine ◽

G. Michael Preston ◽

Kurt F. Weiberth ◽

...

Keyword(s):

Molecular Chaperones ◽

Transmembrane Helix ◽

Integral Membrane Proteins ◽

Membrane Insertion ◽

Transmembrane Helices ◽

Vitro Assay ◽

Dual Pass ◽

Erad Pathway ◽

Insight Into

Integral membrane proteins fold inefficiently and are susceptible to turnover via the endoplasmic reticulum–associated degradation (ERAD) pathway. During ERAD, misfolded proteins are recognized by molecular chaperones, polyubiquitinated, and retrotranslocated to the cytoplasm for proteasomal degradation. Although many aspects of this pathway are defined, how transmembrane helices (TMHs) are removed from the membrane and into the cytoplasm before degradation is poorly understood. In this study, we asked whether the hydrophobic character of a TMH acts as an energetic barrier to retrotranslocation. To this end, we designed a dual-pass model ERAD substrate, Chimera A*, which contains the cytoplasmic misfolded domain from a characterized ERAD substrate, Sterile 6* (Ste6p*). We found that the degradation requirements for Chimera A* and Ste6p* are similar, but Chimera A* was retrotranslocated more efficiently than Ste6p* in an in vitro assay in which retrotranslocation can be quantified. We then constructed a series of Chimera A* variants containing synthetic TMHs with a range of ΔG values for membrane insertion. TMH hydrophobicity correlated inversely with retrotranslocation efficiency, and in all cases, retrotranslocation remained Cdc48p dependent. These findings provide insight into the energetic restrictions on the retrotranslocation reaction, as well as a new computational approach to predict retrotranslocation efficiency.

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Structural basis for membrane insertion by the human ER membrane protein complex

Science ◽

10.1126/science.abb5008 ◽

2020 ◽

pp. eabb5008 ◽

Cited By ~ 7

Author(s):

Tino Pleiner ◽

Giovani Pinton Tomaleri ◽

Kurt Januszyk ◽

Alison J. Inglis ◽

Masami Hazu ◽

...

Keyword(s):

Membrane Protein ◽

Protein Complex ◽

Structural Basis ◽

Membrane Insertion ◽

Transmembrane Helices ◽

Membrane Protein Complex ◽

Cryo Electron Microscopy ◽

Membrane Thinning ◽

Positively Charged ◽

Er Membrane

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit ER membrane protein complex (EMC) is a conserved co- and post-translational insertase at the endoplasmic reticulum. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 Å by cryo-electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer.

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The N-Terminal Region of the Escherichia coli WecA (Rfe) Protein, Containing Three Predicted Transmembrane Helices, Is Required for Function but Not for Membrane Insertion

Journal of Bacteriology ◽

10.1128/jb.182.2.498-503.2000 ◽

2000 ◽

Vol 182 (2) ◽

pp. 498-503 ◽

Cited By ~ 25

Author(s):

Amal O. Amer ◽

Miguel A. Valvano

Keyword(s):

Escherichia Coli ◽

Translation Initiation ◽

Terminal Region ◽

Membrane Localization ◽

Membrane Insertion ◽

Transmembrane Helices

ABSTRACT The correct site for translation initiation for Escherichia coli WecA (Rfe), presumably involved in catalyzing the transfer of N-acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives. The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein.

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Membrane integration and topology of RIFIN and STEVOR proteins of thePlasmodium falciparumparasite

10.1101/653998 ◽

2019 ◽

Cited By ~ 1

Author(s):

Annika Andersson ◽

Renuka Kudva ◽

Anastasia Magoulopoulou ◽

Quentin Lejarre ◽

Patricia Lara ◽

...

Keyword(s):

Vascular Endothelium ◽

Blood Cells ◽

Open Reading Frame ◽

Malarial Parasite ◽

Membrane Insertion ◽

Transmembrane Helices ◽

Reading Frame ◽

And Topology ◽

Infected Cells ◽

Er Membrane

ABSTRACTThe malarial parasitePlasmodium, infects red blood cells by remodeling them and transporting its own proteins to their cell surface. These proteins trigger adhesion of infected cells to uninfected cells (rosetting), and to the vascular endothelium, obstructing blood flow and contributing to pathogenesis. RIFINs (P. falciparum-encoded repetitive interspersed families of polypeptides) and STEVORs (subtelomeric variable open reading frame), are two classes of proteins that are involved in rosetting. Here we study the membrane insertion and topology of three RIFIN and two STEVOR proteins, employing a well-established assay that uses N-linked glycosylation of sites within the protein as a measure to assess the topology a protein adopts when inserted into the ER membrane. Our results indicate that all the proteins tested assume an overall topology of Ncyt-Ccyt, with predicted transmembrane helices TM1 and TM3 integrated into the ER membrane. We also show that the segments predicted as TM2 do not reside in the membrane. Our conclusions are consistent with other recent topology studies on RIFIN and STEVOR proteins.

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Membrane protein insertion and assembly by the bacterial holo-translocon SecYEG–SecDF–YajC–YidC

Biochemical Journal ◽

10.1042/bcj20160545 ◽

2016 ◽

Vol 473 (19) ◽

pp. 3341-3354 ◽

Cited By ~ 31

Author(s):

Joanna Komar ◽

Sara Alvira ◽

Ryan J. Schulze ◽

Remy Martin ◽

Jelger A. Lycklama a Nijeholt ◽

...

Keyword(s):

Membrane Protein ◽

Signal Recognition Particle ◽

Common Mechanism ◽

Membrane Insertion ◽

Transmembrane Helices ◽

Protein Insertion ◽

Wide Range ◽

Membrane Protein Insertion ◽

Bona Fide ◽

Lateral Gate

Protein secretion and membrane insertion occur through the ubiquitous Sec machinery. In this system, insertion involves the targeting of translating ribosomes via the signal recognition particle and its cognate receptor to the SecY (bacteria and archaea)/Sec61 (eukaryotes) translocon. A common mechanism then guides nascent transmembrane helices (TMHs) through the Sec complex, mediated by associated membrane insertion factors. In bacteria, the membrane protein ‘insertase’ YidC ushers TMHs through a lateral gate of SecY to the bilayer. YidC is also thought to incorporate proteins into the membrane independently of SecYEG. Here, we show the bacterial holo-translocon (HTL) — a supercomplex of SecYEG–SecDF–YajC–YidC — is a bona fide resident of the Escherichia coli inner membrane. Moreover, when compared with SecYEG and YidC alone, the HTL is more effective at the insertion and assembly of a wide range of membrane protein substrates, including those hitherto thought to require only YidC.

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ER membrane protein complex is required for the insertions of late-synthesized transmembrane helices of Rh1 in Drosophila photoreceptors

Molecular Biology of the Cell ◽

10.1091/mbc.e19-08-0434 ◽

2019 ◽

Vol 30 (23) ◽

pp. 2890-2900 ◽

Cited By ~ 5

Author(s):

Naoki Hiramatsu ◽

Tatsuya Tago ◽

Takunori Satoh ◽

Akiko K. Satoh

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Protein Complex ◽

Model System ◽

Membrane Insertion ◽

Transmembrane Helices ◽

Membrane Protein Complex ◽

C Terminus ◽

Wide Range ◽

Er Membrane

Most membrane proteins are synthesized on and inserted into the membrane of the endoplasmic reticulum (ER), in eukaryote. The widely conserved ER membrane protein complex (EMC) facilitates the biogenesis of a wide range of membrane proteins. In this study, we investigated the EMC function using Drosophila photoreceptor as a model system. We found that the EMC was necessary only for the biogenesis of a subset of multipass membrane proteins such as rhodopsin (Rh1), TRP, TRPL, Csat, Cni, SERCA, and Na+K+ATPase α, but not for that of secretory or single-pass membrane proteins. Additionally, in EMC-deficient cells, Rh1 was translated to its C terminus but degraded independently from ER-associated degradation. Thus, EMC exerted its effect after translation but before or during the membrane integration of transmembrane domains (TMDs). Finally, we found that EMC was not required for the stable expression of the first three TMDs of Rh1 but was required for that of the fourth and fifth TMDs. Our results suggested that EMC is required for the ER membrane insertion of succeeding TMDs of multipass membrane proteins.

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Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices

10.1101/2021.12.23.474019 ◽

2021 ◽

Author(s):

Felix Nicolaus ◽

Fatima Ibrahimi ◽

Anne den Besten ◽

Gunnar von Heijne

Keyword(s):

Hydrophobic Interactions ◽

Protein A ◽

Membrane Insertion ◽

Transmembrane Helices ◽

Hydrophobic Residues ◽

Electrostatic And Hydrophobic Interactions ◽

Sequence Elements ◽

Critical Residues ◽

First Contact ◽

Positively Charged

During SecYEG-mediated cotranslational insertion of membrane proteins, transmembrane helices (TMHs) first make contact with the membrane when their N-terminal end is ~45 residues away from the peptidyl transferase center. However, we recently uncovered instances where the first contact is delayed by up to ~10 residues. Here, we recapitulate these effects using a model TMH fused to two short segments from the BtuC protein: a positively charged loop and a re-entrant loop. We show that the critical residues are two Arg residues in the positively charged loop and four hydrophobic residues in the re-entrant loop. Thus, both electrostatic and hydrophobic interactions involving sequence elements that are not part of a TMH can impact the way the latter behaves during membrane insertion.

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