A trap mutant reveals the physiological client spectrum of TRC40

TY - JOUR

T1 - A trap mutant reveals the physiological client spectrum of TRC40

AU - Coy-Vergara, Javier

AU - Rivera-Monroy, Jhon

AU - Urlaub, Henning

AU - Lenz, Christof

AU - Schwappach, Blanche

N1 - © 2019. Published by The Company of Biologists Ltd.

PY - 2019/7/1

Y1 - 2019/7/1

N2 - The transmembrane recognition complex (TRC) pathway targets tail-anchored (TA) proteins to the membrane of the endoplasmic reticulum (ER). While many TA proteins are known to be able to use this pathway, it is essential for the targeting of only a few. Here, we uncover a large number of TA proteins that engage with TRC40 when other targeting machineries are fully operational. We use a dominant-negative ATPase-impaired mutant of TRC40 in which aspartate 74 was replaced by a glutamate residue to trap TA proteins in the cytoplasm. Manipulation of the hydrophobic TA-binding groove in TRC40 (also known as ASNA1) reduces interaction with most, but not all, substrates suggesting that co-purification may also reflect interactions unrelated to precursor protein targeting. We confirm known TRC40 substrates and identify many additional TA proteins interacting with TRC40. By using the trap approach in combination with quantitative mass spectrometry, we show that Golgi-resident TA proteins such as the golgins golgin-84, CASP and giantin as well as the vesicle-associated membrane-protein-associated proteins VAPA and VAPB interact with TRC40. Thus, our results provide new avenues to assess the essential role of TRC40 in metazoan organisms.This article has an associated First Person interview with the first author of the paper.

AB - The transmembrane recognition complex (TRC) pathway targets tail-anchored (TA) proteins to the membrane of the endoplasmic reticulum (ER). While many TA proteins are known to be able to use this pathway, it is essential for the targeting of only a few. Here, we uncover a large number of TA proteins that engage with TRC40 when other targeting machineries are fully operational. We use a dominant-negative ATPase-impaired mutant of TRC40 in which aspartate 74 was replaced by a glutamate residue to trap TA proteins in the cytoplasm. Manipulation of the hydrophobic TA-binding groove in TRC40 (also known as ASNA1) reduces interaction with most, but not all, substrates suggesting that co-purification may also reflect interactions unrelated to precursor protein targeting. We confirm known TRC40 substrates and identify many additional TA proteins interacting with TRC40. By using the trap approach in combination with quantitative mass spectrometry, we show that Golgi-resident TA proteins such as the golgins golgin-84, CASP and giantin as well as the vesicle-associated membrane-protein-associated proteins VAPA and VAPB interact with TRC40. Thus, our results provide new avenues to assess the essential role of TRC40 in metazoan organisms.This article has an associated First Person interview with the first author of the paper.

KW - Arsenite Transporting ATPases/genetics

KW - Cytoplasm/metabolism

KW - Gene Silencing

KW - HeLa Cells

KW - Humans

KW - Hydrophobic and Hydrophilic Interactions

KW - Models, Biological

KW - Mutation/genetics

KW - Protein Binding

KW - Subcellular Fractions/metabolism

KW - Substrate Specificity

U2 - 10.1242/jcs.230094

DO - 10.1242/jcs.230094

M3 - SCORING: Journal article

C2 - 31182645

VL - 132

JO - J CELL SCI

JF - J CELL SCI

SN - 0021-9533

IS - 13

ER -