Multiple selection filters ensure accurate tail-anchored membrane protein targeting

Rao, Meera; Okreglak, Voytek; Chio, Un Seng; Cho, Hyunjun; Walter, Peter; Shan, Shu‐ou

doi:10.7554/elife.21301

Cited by 85 publications

(193 citation statements)

References 64 publications

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“…1B). Fluorophores were incorporated at a nonconserved loop in the Get3 helical domain, and labeling does not affect the activity of Get3 (12). We used confocal microscopy with alternating laser excitation with microsecond time resolution (μs-ALEX) to detect and quantify the fluorescence of single molecules transiting through a femtoliter-scale observation volume, and extracted relative FRET efficiencies (E*) for individual molecules (22) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To determine the conformation of Get3 when bound to the TA substrate, we assembled Get3•TA complexes by in vitro translation of Bos1, a model GET substrate (12), in Escherichia coli lysate in the presence of Get3 and affinity-purified Get3•TA complexes via the 3xStrep-tag on Bos1 (12) (Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

“…S4B). The lower limit for the lifetime of other Get3•TA complexes was 35-60 min (12). Thus, TAs are stably bound to Get3 despite the large-scale conformational fluctuations of Get3.…”

Section: -H)mentioning

confidence: 97%

“…1B). Closed ATP• Get3 is preferentially bound by the Get4/5 complex (9,10), which bridges between Get3 and the upstream cochaperone Sgt2 and stimulates TA transfer from Sgt2 onto Get3 (11)(12)(13). After dissociation from Get4/5, the Get3•TA complex hydrolyzes ATP and interacts with the Get1/2 membrane receptors (10,14).…”

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confidence: 99%

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A protean clamp guides membrane targeting of tail-anchored proteins

Chio

Chung

Weiss

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Proper localization of proteins to target membranes is a fundamental cellular process. How the nature and dynamics of the targeting complex help guide substrate proteins to the target membrane is not understood for most pathways. Here, we address this question for the conserved ATPase guided entry of tail-anchored protein 3 (Get3), which targets the essential class of tail-anchored proteins (TAs) to the endoplasmic reticulum (ER). Single-molecule fluorescence spectroscopy showed that, contrary to previous models of a static closed Get3•TA complex, Get3 samples open conformations on the submillisecond timescale upon TA binding, generating a fluctuating "protean clamp" that stably traps the substrate. Point mutations at the ATPase site bias Get3 toward closed conformations, uncouple TA binding from induced Get3•Get4/5 disassembly, and inhibit the ER targeting of the Get3•TA complex. These results demonstrate an essential role of substrate-induced Get3 dynamics in driving TA targeting to the membrane, and reveal a tightly coupled channel of communication between the TA-binding site, ATPase site, and effector interaction surfaces of Get3. Our results provide a precedent for large-scale dynamics in a substrate-bound chaperone, which provides an effective mechanism to retain substrate proteins with high affinity while also generating functional switches to drive vectorial cellular processes.protein targeting | chaperones | protein dynamics | single-molecule spectroscopy | ATPases O ver 35% of proteins need to be localized to the correct cellular destinations after their initial synthesis in the cytosol. These protein-targeting processes are essential for the establishment and maintenance of compartmentalization in all cells and pose complex mechanistic challenges to targeting machineries. To minimize improper exposure of substrate proteins in the cytosol, targeting factors must bind substrate proteins with high stability. This requirement is especially stringent during the targeting of integral membrane proteins, whose high aggregation propensity in the cytosol and other aqueous cellular environments demands that targeting factors also serve as effective chaperones to protect substrates from aggregation. Further, to minimize futile cycling of targeting factors, loading of substrates on the targeting factor must be tightly coupled to their delivery to membrane receptor sites. Finally, once at the target membrane, the targeting machinery must readily switch to a lowaffinity state to release substrate proteins to receptor complexes, translocases, or the phospholipid bilayer. With a few exceptions (1, 2), the nature and dynamics of protein targeting complexes and how their biophysical properties help meet these complex functional demands are not well understood, especially for posttranslational protein targeting pathways.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…S4B). The lower limit for the lifetime of other Get3•TA complexes was 35-60 min (12). Thus, TAs are stably bound to Get3 despite the large-scale conformational fluctuations of Get3.…”

Section: -H)mentioning

confidence: 97%

mentioning

confidence: 99%

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A protean clamp guides membrane targeting of tail-anchored proteins

Chio

Chung

Weiss

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…It was recently noted that multiple selection filters have been used to distinguish correct and incorrect substrates based on minor differences of the TMD and C-terminal elements of TA proteins (9). Because several TA proteins have been identified as the AtGET3a substrates, future studies will aim at elucidating the underlying molecular mechanisms for plant TA protein recognition in plants to understand why and how the plant GET complex impacts the substrates to different extents (e.g., abundance or mislocalization), and their other specific physiological roles during plant growth and development.…”

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confidence: 99%

Targeting tail-anchored proteins into plant organelles

Zhuang¹,

Chung²,

Jiang³

2017

Proc. Natl. Acad. Sci. U.S.A.

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In vitro Assays for Targeting and Insertion of Tail‐Anchored Proteins Into the ER Membrane

2018

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Membrane proteins mediate numerous essential cellular functions. Due to the aggregation propensity of hydrophobic transmembrane domains in aqueous environments, the targeting and insertion of membrane proteins pose major challenges to cells. In the Guided Entry of Tail-anchored protein (GET) pathway, an essential class of newly synthesized tail-anchored proteins (TAs) are chaperoned and guided by multiple targeting factors to the endoplasmic reticulum (ER) membrane. Deciphering the molecular mechanism of this cellular process has benefitted from successful in vitro reconstitution of individual molecular events in the GET pathway with purified components. Here we describe recently developed protocols for in vitro reconstitution of functional complexes of TA substrates with their targeting factors, for monitoring the transfer of TAs between targeting factors, and for the insertion of TA into the microsomal membrane. These procedures are generally applicable to the interrogation of other post-translational membrane protein targeting pathways.

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Multiple selection filters ensure accurate tail-anchored membrane protein targeting

Cited by 85 publications

References 64 publications

A protean clamp guides membrane targeting of tail-anchored proteins

A protean clamp guides membrane targeting of tail-anchored proteins

Targeting tail-anchored proteins into plant organelles

In vitro Assays for Targeting and Insertion of Tail‐Anchored Proteins Into the ER Membrane

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