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Abstract

The 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 substituted by glutamate to trap TA-proteins in the cytoplasm. Manipulation of TRC40's hydrophobic TA-binding groove 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. 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 tackle the essential role of TRC40 in metazoan organisms.