Publication Date:
2014-07-22
Description:
Hundreds of tail-anchored proteins, including soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs) involved in vesicle fusion, are inserted post-translationally into the endoplasmic reticulum membrane by a dedicated protein-targeting pathway. Before insertion, the carboxy-terminal transmembrane domains of tail-anchored proteins are shielded in the cytosol by the conserved targeting factor Get3 (in yeast; TRC40 in mammals). The Get3 endoplasmic-reticulum receptor comprises the cytosolic domains of the Get1/2 (WRB/CAML) transmembrane complex, which interact individually with the targeting factor to drive a conformational change that enables substrate release and, as a consequence, insertion. Because tail-anchored protein insertion is not associated with significant translocation of hydrophilic protein sequences across the membrane, it remains possible that Get1/2 cytosolic domains are sufficient to place Get3 in proximity with the endoplasmic-reticulum lipid bilayer and permit spontaneous insertion to occur. Here we use cell reporters and biochemical reconstitution to define mutations in the Get1/2 transmembrane domain that disrupt tail-anchored protein insertion without interfering with Get1/2 cytosolic domain function. These mutations reveal a novel Get1/2 insertase function, in the absence of which substrates stay bound to Get3 despite their proximity to the lipid bilayer; as a consequence, the notion of spontaneous transmembrane domain insertion is a non sequitur. Instead, the Get1/2 transmembrane domain helps to release substrates from Get3 by capturing their transmembrane domains, and these transmembrane interactions define a bona fide pre-integrated intermediate along a facilitated route for tail-anchor entry into the lipid bilayer. Our work sheds light on the fundamental point of convergence between co-translational and post-translational endoplasmic-reticulum membrane protein targeting and insertion: a mechanism for reducing the ability of a targeting factor to shield its substrates enables substrate handover to a transmembrane-domain-docking site embedded in the endoplasmic-reticulum membrane.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342754/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342754/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Wang, Fei -- Chan, Charlene -- Weir, Nicholas R -- Denic, Vladimir -- R01 GM099943/GM/NIGMS NIH HHS/ -- R01GM0999943-01/GM/NIGMS NIH HHS/ -- England -- Nature. 2014 Aug 28;512(7515):441-4. doi: 10.1038/nature13471. Epub 2014 Jul 20.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cellular Biology, Harvard University, Northwest Labs, Cambridge, Massachusetts 02138, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25043001" target="_blank"〉PubMed〈/a〉
Keywords:
Adaptor Proteins, Vesicular Transport/chemistry/genetics/*metabolism
;
Adenosine Triphosphatases/metabolism
;
Binding Sites
;
Endoplasmic Reticulum/chemistry/enzymology/*metabolism
;
Guanine Nucleotide Exchange Factors/metabolism
;
Intracellular Membranes/chemistry/enzymology/*metabolism
;
Lipid Bilayers/chemistry/metabolism
;
Membrane Proteins/chemistry/genetics/*metabolism
;
Multiprotein Complexes/chemistry/*metabolism
;
Mutant Proteins/chemistry/genetics/metabolism
;
Mutation
;
Protein Binding
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Protein Structure, Tertiary/genetics
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Protein Transport/genetics
;
Saccharomyces cerevisiae/*cytology/*enzymology/genetics/metabolism
;
Saccharomyces cerevisiae Proteins/chemistry/genetics/*metabolism
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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