mRNA encoding Sec61β, a tail-anchored protein, is localized on the endoplasmic reticulum

Abstract

Although 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.

Single-Molecule Quantification of Translation-Dependent Association of mRNAs with the Endoplasmic Reticulum

“…Transferrin receptor (TFRC) transcripts, which encode a membrane protein, were resistant to extraction (71%) and, thus, likely to be ER associated ( Figure 1B). This is similar to what was previously found for other mRNAs encoding membrane-bound and secretory proteins (Cui et al, , 2015Jagannathan et al, 2014). In contrast, only a minority of mRNAs encoding cytosolic or nuclear proteins-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), DnaJ heat-shock protein family (Hsp40) member B4 (DNAJB4), forkhead box K2 (FOXK2) transcription factor, and 5 0 -nucleotidase domain containing protein 2 (5NTDC2)-was resistant to digitonin extraction.…”

Discrimination between the endoplasmic reticulum and mitochondria by spontaneously inserting tail‐anchored proteins

“…To explain the discrepancy between the precise in vivo targeting of b5‐ER and b5‐RR and their promiscuous behavior in cell‐free systems, we considered the possibility that the capacity of the 2 forms to discriminate between the ER and the MOM might depend on a segregated localization of their mRNAs, as reported for Sec61β, or on ribosome‐associated factors, which would capture the substrate immediately after the release from the ribosome . If either of these mechanisms were operating, the purified recombinant proteins, microinjected into the cytosol of cultured cells, would be incapable of correct targeting.…”

“…Another potential mechanism could be contributed by mRNA localization . In the case of TA proteins, Cui et al reported that the mRNAs coding for Sec61β and for nesprin are partially localized to the ER, thus facilitating the subsequent insertion of the translated polypeptides. Finally, the precision of TA protein localization can be fine‐tuned by postinsertional quality control mechanisms, as exemplified by the AAA ATPase Msp1/ATAD, which extracts mislocalized TA proteins from the MOM for subsequent degradation …”

mRNA localization as a rheostat to regulate subcellular gene expression

“…This might especially be true in cases where the mRNA may localize right near its translational product, especially for many organelle‐targeted transcripts. For example, Sec61β mRNA, which encodes a tail‐anchored protein that is a component of the translocon (the main protein‐conducting pore of the ER), localizes to the ER membrane . This is surprising, since tail‐anchored proteins are thought to be inserted into membranes post‐translationally placing their mRNAs in the cytosol .…”

“…This is surprising, since tail‐anchored proteins are thought to be inserted into membranes post‐translationally placing their mRNAs in the cytosol . Interestingly, the overexpression of GFP‐Sec61β evicts other mRNAs from the ER surface, probably because its mRNA saturates all the mRNA‐binding sites on this organelle (i.e., ribosomes that are docked onto the translocon) . It is possible that the endogenous Sec61β mRNA is translated only when there is an excess of translocon that lacks its encoded protein Sec61β.…”