scholarly journals Decision letter: Structure of the AAA protein Msp1 reveals mechanism of mislocalized membrane protein extraction

2020 ◽  
Author(s):  
Peter Chien ◽  
Gabriel C Lander
Keyword(s):  
Membrane Protein ◽  
Protein Extraction ◽  
Aaa Protein

scholarly journals Structure of the AAA protein Msp1 reveals mechanism of mislocalized membrane protein extraction

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lan Wang ◽  
Alexander Myasnikov ◽  
Xingjie Pan ◽  
Peter Walter
Keyword(s):  
Membrane Protein ◽  
Atp Hydrolysis ◽  
Protein Extraction ◽  
Aromatic Amino Acids ◽  
Extraction Process ◽  
Comprehensive Model ◽  
Unique Role ◽  
Cell Organization ◽  
Architectural Principles ◽  
Aaa Protein

The AAA protein Msp1 extracts mislocalized tail-anchored membrane proteins and targets them for degradation, thus maintaining proper cell organization. How Msp1 selects its substrates and firmly engages them during the energetically unfavorable extraction process remains a mystery. To address this question, we solved cryo-EM structures of Msp1-substrate complexes at near-atomic resolution. Akin to other AAA proteins, Msp1 forms hexameric spirals that translocate substrates through a central pore. A singular hydrophobic substrate recruitment site is exposed at the spiral’s seam, which we propose positions the substrate for entry into the pore. There, a tight web of aromatic amino acids grips the substrate in a sequence-promiscuous, hydrophobic milieu. Elements at the intersubunit interfaces coordinate ATP hydrolysis with the subunits’ positions in the spiral. We present a comprehensive model of Msp1’s mechanism, which follows general architectural principles established for other AAA proteins yet specializes Msp1 for its unique role in membrane protein extraction.

scholarly journals Selective Enrichment of Membrane Proteins by Partition Phase Separation for Proteomic Studies

2003 ◽  
Vol 2003 (4) ◽  
pp. 249-255 ◽  
Author(s):  
M. Walid Qoronfleh ◽  
Betsy Benton ◽  
Ray Ignacio ◽  
Barbara Kaboord
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Extraction ◽  
Yeast Cells ◽  
Protein Markers ◽  
Two Phase ◽  
Western Blots ◽  
Selective Enrichment ◽  
Phase Partitioning ◽  
Hydrophobic Fraction

The human proteome project will demand faster, easier, and more reliable methods to isolate and purify protein targets. Membrane proteins are the most valuable group of proteins since they are the target for 70–80% of all drugs. Perbio Science has developed a protocol for the quick, easy, and reproducible isolation of integral membrane proteins from eukaryotic cells. This procedure utilizes a proprietary formulation to facilitate cell membrane disruption in a mild, nondenaturing environment and efficiently solubilizes membrane proteins. The technique utilizes a two-phase partitioning system that enables the class separation of hydrophobic and hydrophilic proteins. A variety of protein markers were used to investigate the partitioning efficiency of the membrane protein extraction reagents (Mem-PER) (Mem-PER is a registered trademark of Pierce Biotechnology, Inc) system. These included membrane proteins with one or more transmembrane spanning domains as well as peripheral and cytosolic proteins. Based on densitometry analyses of our Western blots, we obtained excellent solubilization of membrane proteins with less than 10% contamination of the hydrophobic fraction with hydrophilic proteins. Compared to other methodologies for membrane protein solubilization that use time-consuming protocols or expensive and cumbersome instrumentation, the Mem-PER reagents system for eukaryotic membrane protein extraction offers an easy, efficient, and reproducible method to isolate membrane proteins from mammalian and yeast cells.

scholarly journals Doa10 is a membrane protein retrotranslocase in ER-associated protein degradation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Claudia C Schmidt ◽  
Vedran Vasic ◽  
Alexander Stein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
Protein Extraction ◽  
Phospholipid Bilayer ◽  
Luminal Side ◽  
Unfolded State ◽  
Membrane Bound ◽  
Reconstituted System

In endoplasmic reticulum-associated protein degradation (ERAD), membrane proteins are ubiquitinated, extracted from the membrane, and degraded by the proteasome. The cytosolic ATPase Cdc48 drives extraction by pulling on polyubiquitinated substrates. How hydrophobic transmembrane (TM) segments are moved from the phospholipid bilayer into cytosol, often together with hydrophilic and folded ER luminal protein parts, is not known. Using a reconstituted system with purified proteins from Saccharomyces cerevisiae, we show that the ubiquitin ligase Doa10 (Teb-4/MARCH6 in animals) is a retrotranslocase that facilitates membrane protein extraction. A substrate’s TM segment interacts with the membrane-embedded domain of Doa10 and then passively moves into the aqueous phase. Luminal substrate segments cross the membrane in an unfolded state. Their unfolding occurs on the luminal side of the membrane by cytoplasmic Cdc48 action. Our results reveal how a membrane-bound retrotranslocase cooperates with the Cdc48 ATPase in membrane protein extraction.

scholarly journals Membrane protein extraction and purification using partially-esterified SMA polymers

2021 ◽  
pp. 183758
Author(s):  
Olivia P. Hawkins ◽  
Christine Parisa T. Jahromi ◽  
Aiman A. Gulamhussein ◽  
Stephanie Nestorow ◽  
Taranpreet Bahra ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Extraction ◽  
Extraction And Purification

Hydrogenated Diglucose Detergents for Membrane-Protein Extraction and Stabilization

Langmuir ◽  
2019 ◽  
Vol 35 (12) ◽  
pp. 4287-4295 ◽  
Author(s):  
Pierre Guillet ◽  
Florian Mahler ◽  
Kelly Garnier ◽  
Gildas Nyame Mendendy Boussambe ◽  
Sébastien Igonet ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Extraction

scholarly journals Membrane protein extraction and purification using styrene–maleic acid (SMA) copolymer: effect of variations in polymer structure

2016 ◽  
Vol 473 (23) ◽  
pp. 4349-4360 ◽  
Author(s):  
Kerrie A. Morrison ◽  
Aneel Akram ◽  
Ashlyn Mathews ◽  
Zoeya A. Khan ◽  
Jaimin H. Patel ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Maleic Acid ◽  
Protein Extraction ◽  
Affinity Purification ◽  
Expression System ◽  
Transmembrane Proteins ◽  
Membrane Extraction ◽  
Total Size ◽  
Average Molecular Mass

The use of styrene–maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5–10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.

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