Abstract
ABSTRACTAlthough one pathway for the post-translational targeting of tail-anchored proteins to the endoplasmic reticulum (ER) has been well defined, it is unclear whether additional pathways exist. Here, we provide evidence that a subset of mRNAs encoding tail-anchored proteins, including Sec61β and nesprin-2, is partially localized to the surface of the ER in mammalian cells. In particular, Sec61b mRNA can be targeted to, and later maintained on, the ER using both translation-dependent and -independent mechanisms. Our data suggests that this process is independent of p180 (also known as RRBP1), a known mRNA receptor on the ER, and the transmembrane domain recognition complex (TRC) pathway components, TRC40 (also known as ASNA1) and BAT3 (also known as BAG6). In addition, our data indicates that Sec61b mRNA might access translocon-bound ribosomes. Our results show that certain tail-anchored proteins are likely to be synthesized directly on the ER, and this facilitates their membrane insertion. Thus, it is clear that mammalian cells utilize multiple mechanisms to ensure efficient targeting of tail-anchored proteins to the surface of the ER.
Highlights
One major mechanism that directs proteins to their correct subcellular destination is localization of their mRNA (Holt and Bullock, 2009; Martin and Ephrussi, 2009)
Sec61b mRNA is partially localized on the endoplasmic reticulum (ER) It is currently believed that mRNAs encoding tail-anchored proteins are first translated by free ribosomes, and that the encoded polypeptide is later post-translationally targeted to the ER through the transmembrane domain recognition complex (TRC) pathway (Rabu et al, 2009; Borgese and Fasana, 2011; Hegde and Keenan, 2011)
To test whether the localization of Sec61b mRNA was translation dependent, we examined the mRNA localization in cells treated with either homoharringtonine (HHT), or with puromycin followed by extraction with EDTA (Puro+EDTA), two treatments that effectively dissociate ribosomes from mRNA (Cui et al, 2012)
Summary
One major mechanism that directs proteins to their correct subcellular destination is localization of their mRNA (Holt and Bullock, 2009; Martin and Ephrussi, 2009). The most widespread example is the localization of mRNAs encoding membrane and secreted proteins to the surface of the ER in eukaryotic cells. This localization facilitates the targeting of the encoded proteins to the secretory pathway (Cui and Palazzo, 2014). It was thought that these mRNAs are exclusively targeted to the ER by their encoded proteins. During their translation, newly synthesized hydrophobic signal sequences or transmembrane domains (TMDs) are recognized as they emerge from the ribosome by the signal recognition particle (SRP), which redirects the mRNA–ribosome–nascent-chain complex to the ER surface.
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