A trap mutant reveals the physiological client spectrum of TRC40
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A trap mutant reveals the physiological client spectrum of TRC40. / Coy-Vergara, Javier; Rivera-Monroy, Jhon; Urlaub, Henning; Lenz, Christof; Schwappach, Blanche.
In: J CELL SCI, Vol. 132, No. 13, 01.07.2019.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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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 -