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1.
Cell Rep ; 20(10): 2287-2293, 2017 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-28877464

RESUMO

The Get1/2 transmembrane complex drives the insertion of tail-anchored (TA) proteins from the cytosolic chaperone Get3 into the endoplasmic reticulum membrane. Mechanistic insight into how Get1/2 coordinates this process is confounded by a lack of understanding of the basic architecture of the complex. Here, we define the oligomeric state of full-length Get1/2 in reconstituted lipid bilayers by combining single-molecule and bulk fluorescence measurements with quantitative in vitro insertion analysis. We show that a single Get1/2 heterodimer is sufficient for insertion and demonstrate that the conserved cytosolic regions of Get1 and Get2 bind asymmetrically to opposing subunits of the Get3 homodimer. Altogether, our results define a simplified model for how Get1/2 and Get3 coordinate TA protein insertion.


Assuntos
Bicamadas Lipídicas/química , Animais , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Retículo Endoplasmático/metabolismo , Hidrólise , Proteínas de Membrana/metabolismo , Ligação Proteica , Multimerização Proteica
2.
Plant Physiol ; 173(2): 1137-1145, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27923985

RESUMO

The Arabidopsis (Arabidopsis thaliana) genome encodes homologs of the Guided Entry of Tail (GET)-anchored protein system for the posttranslational insertion of tail-anchored (TA) proteins into endoplasmic reticulum (ER) membranes. In yeast, TA proteins are loaded onto the cytosolic targeting factor Get3 and are then delivered to the membrane-associated Get1/2 complex for insertion into ER membranes. The role of the GET system in Arabidopsis was investigated by monitoring the membrane insertion of a tail-anchored protein, SYP72, a syntaxin. SYP72 bound to yeast Get3 in vitro, forming a Get3-SYP72 fusion complex that could be inserted into yeast GET1/2-containing proteoliposomes. The Arabidopsis GET system functioned in vivo to insert TA proteins into ER membranes as demonstrated by the fact that the YFP-tagged SYP72 localized to the ER in wild-type plants but accumulated as cytoplasmic inclusions in get1, get3, or get4 mutants. The GET mutants get1 and get3 were less tolerant of ER stress agents and showed symptoms of ER stress even under unstressed conditions. Hence, the GET system is responsible for the insertion of TA proteins into the ER in Arabidopsis, and mutants with GET dysfunctions are more susceptible to ER stress.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Qa-SNARE/metabolismo , Mutação/genética , Fenótipo , Proteolipídeos/metabolismo , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Especificidade por Substrato
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