scholarly journals Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Assaf Elazar ◽  
Jonathan Weinstein ◽  
Ido Biran ◽  
Yearit Fridman ◽  
Eitan Bibi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Point Mutations ◽  
Scanning Method ◽  
Strongly Coupled ◽  
Protein Insertion ◽  
Membrane Protein Insertion ◽  
Self Association ◽  
Reliable Quantification ◽  
Natural Amino Acids

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

scholarly journals Isolation of Cold-Sensitive yidC Mutants Provides Insights into the Substrate Profile of the YidC Insertase and the Importance of Transmembrane 3 in YidC Function

2007 ◽  
Vol 189 (24) ◽  
pp. 8961-8972 ◽  
Author(s):  
Jijun Yuan ◽  
Gregory J. Phillips ◽  
Ross E. Dalbey
Keyword(s):  
Membrane Protein ◽  
Expression System ◽  
Point Mutations ◽  
Integral Membrane Protein ◽  
Cold Sensitivity ◽  
Membrane Insertion ◽  
Protein Insertion ◽  
Amino Terminal ◽  
Membrane Protein Insertion ◽  
Cold Sensitive

ABSTRACT YidC, a 60-kDa integral membrane protein, plays an important role in membrane protein insertion in bacteria. YidC can function together with the SecYEG machinery or operate independently as a membrane protein insertase. In this paper, we describe two new yidC mutants that lead to a cold-sensitive phenotype in bacterial cell growth. Both alleles impart a cold-sensitive phenotype and result from point mutations localized to the third transmembrane (TM3) segment of YidC, indicating that this region is crucial for YidC function. We found that the yidC(C423R) mutant confers a weak phenotype on membrane protein insertion while a yidC(P431L) mutant leads to a stronger phenotype. In both cases, the affected substrates include the Pf3 coat protein and ATP synthase F1Fo subunit c (FoC), while CyoA (the quinol binding subunit of the cytochrome bo3 quinol oxidase complex) and wild-type procoat are slightly affected or not affected in either cold-sensitive mutant. To determine if the different substrates require various levels of YidC activity for membrane insertion, we performed studies where YidC was depleted using an arabinose-dependent expression system. We found that −3M-PC-Lep (a construct with three negatively charged residues inserted into the middle of the procoat-Lep [PC-Lep] protein) and Pf3 P2 (a construct with the Lep P2 domain added at the C terminus of Pf3 coat) required the highest amount of YidC and that CyoA-N-P2 (a construct with the amino-terminal part of CyoA fused to the Lep P2 soluble domain) and PC-Lep required the least, while FoC required moderate YidC levels. Although the cold-sensitive mutations can preferentially affect one substrate over another, our results indicate that different substrates require different levels of YidC activity for membrane insertion. Finally, we obtained several intragenic suppressors that overcame the cold sensitivity of the C423R mutation. One pair of mutations suggests an interaction between TM2 and TM3 of YidC. The studies reveal the critical regions of the YidC protein and provide insight into the substrate profile of the YidC insertase.

Structure of YidC from Thermotoga maritima and its implications for YidC‐mediated membrane protein insertion

2018 ◽  
Vol 32 (5) ◽  
pp. 2411-2421 ◽  
Author(s):  
Yanlong Xin ◽  
Yan Zhao ◽  
Jiangge Zheng ◽  
Haizhen Zhou ◽  
Xuejun Cai Zhang ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Thermotoga Maritima ◽  
Protein Insertion ◽  
Membrane Protein Insertion

Membrane protein import in yeast mitochondria

2000 ◽  
Vol 28 (4) ◽  
pp. 495-499 ◽  
Author(s):  
K. Tokatlidis ◽  
S. Vial ◽  
P. Luciano ◽  
M. Vergnolle ◽  
S. Clémence
Keyword(s):  
Membrane Protein ◽  
Molecular Mechanism ◽  
Protein Import ◽  
Mitochondrial Matrix ◽  
Yeast Mitochondria ◽  
Inner Membrane ◽  
Protein Insertion ◽  
The Past ◽  
Membrane Protein Insertion ◽  
Membrane Bound

The protein import pathway that targets proteins to the mitochondrial matrix has been extensively characterized in the past 15 years. Variations of this import pathway account for the sorting of proteins to other compartments as well, but the insertion of integral inner membrane proteins lacking a presequence is mediated by distinct translocation machinery. This consists of a complex of Tim9 and Tim10, two homologous, Zn2+-binding proteins that chaperone the passage of the hydrophobic precursor across the aqueous inter-membrane space. The precursor is then targeted to another, inner-membrane-bound, complex of at least five subunits that facilitates insertion. Biochemical and genetic experiments have identified the key components of this process; we are now starting to understand the molecular mechanism. This review highlights recent advances in this new membrane protein insertion pathway.

scholarly journals Membrane Protein Insertion into and Compatibility with Biomimetic Membranes

2017 ◽  
Vol 1 (7) ◽  
pp. 1700053 ◽  
Author(s):  
Tingwei Ren ◽  
Mustafa Erbakan ◽  
Yuexiao Shen ◽  
Eduardo Barbieri ◽  
Patrick Saboe ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Insertion ◽  
Biomimetic Membranes ◽  
Membrane Protein Insertion
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