Abstract
ABSTRACTTail-anchored (TA) proteins are transmembrane proteins with a single C-terminal transmembrane domain, which functions as both their subcellular targeting signal and membrane anchor. We show that knockout of TRC40 in cultured human cells has a relatively minor effect on endogenous TA proteins, despite their apparent reliance on this pathway in vitro. These findings support recent evidence that the canonical TRC40 pathway is not essential for TA protein biogenesis in vivo. We therefore investigated the possibility that other ER-targeting routes can complement the TRC40 pathway and identified roles for both the SRP pathway and the recently described mammalian SND pathway in TA protein biogenesis. We conclude that, although TRC40 normally plays an important role in TA protein biogenesis, it is not essential, and speculate that alternative pathways for TA protein biogenesis, including those identified in this study, contribute to the redundancy of the TRC40 pathway.
Highlights
Targeting of membrane and secretory proteins to the mammalian endoplasmic reticulum (ER) can occur through either a cotranslational signal recognition particle (SRP)-dependent pathway (Nyathi et al, 2013) or distinct post-translational pathways (Johnson et al, 2013)
Deletion of TRC40 has differential effects on endogenous TA proteins Previous studies of the contribution of the TRC40 pathway to TA protein biogenesis in metazoans have relied upon the perturbation of the WRB–CAML receptor complex (Daniele et al, 2016; Lin et al, 2016; Rivera-Monroy et al, 2016; Vogl et al, 2016)
In order to determine the effect of the TRC40 knockout on TA protein biogenesis, we analysed the steady-state levels of three endogenous TA proteins, all of which have previously been suggested to utilise the TRC40 pathway (Favaloro et al, 2008, 2010; Rivera-Monroy et al, 2016)
Summary
Targeting of membrane and secretory proteins to the mammalian endoplasmic reticulum (ER) can occur through either a cotranslational SRP-dependent pathway (Nyathi et al, 2013) or distinct post-translational pathways (Johnson et al, 2013). In both cases, the binding of specific cytosolic targeting factors to hydrophobic signal sequences or transmembrane domains prevents their exposure and thereby minimises subsequent inappropriate interactions that might lead to their aggregation (Cross et al, 2009). The mammalian cytosolic ATPase TRC40 ( known as ASNA1 and Get3) was first identified as an ER-targeting factor that binds post-translationally to the C-terminal hydrophobic domains of TA proteins and facilitates. Once a TA protein cargo is loaded onto TRC40, a heterodimeric ER membrane receptor complex (WRB–CAML) facilitates its insertion (Vilardi et al, 2011; Wang et al, 2014; Yamamoto and Sakisaka, 2012)
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