Role of Protein Translocation Pathways across the Endoplasmic Reticulum in Trypanosoma brucei

Abstract

The translocation of secretory and membrane proteins across the endoplasmic reticulum (ER) membrane is mediated by co-translational (via the signal recognition particle (SRP)) and post-translational mechanisms. In this study, we investigated the relative contributions of these two pathways in trypanosomes. A homologue of SEC71, which functions in the post-translocation chaperone pathway in yeast, was identified and silenced by RNA interference. This factor is essential for parasite viability. In SEC71-silenced cells, signal peptide (SP)-containing proteins traversed the ER, but several were mislocalized, whereas polytopic membrane protein biogenesis was unaffected. Surprisingly trypanosomes can interchangeably utilize two of the pathways to translocate SPcontaining proteins except for glycosylphosphatidylinositol-anchored proteins, whose level was reduced in SEC71-silenced cells but not in cells depleted for SRP68, an SRP-binding protein. Entry of SP-containing proteins to the ER was significantly blocked only in cells co-silenced for the two translocation pathways (SEC71 and SRP68). SEC63, a factor essential for both translocation pathways in yeast, was identified and silenced by RNA interference. SEC63 silencing affected entry to the ER of both SP-containing proteins and polytopic membrane proteins, suggesting that, as in yeast, this factor is essential for both translocation pathways in vivo. This study suggests that, unlike bacteria or other eukaryotes, trypanosomes are generally promiscuous in their choice of mechanism for translocating SPcontaining proteins to the ER, although the SRP-independent pathway is favored for glycosylphosphatidylinositol-anchored proteins, which are the most abundant surface proteins in these parasites.

Post-translational import of protein into the endoplasmic reticulum of a trypanosome: an in vitro system for discovery of anti-trypanosomal chemical entities

“…Thirdly, we demonstrate post-translational import of VSG_117 into TbRM in vitro . Gene knockdown experiments in vivo [4,43] and in vitro microsomal studies of protein import into the ER of T. brucei (the present study) complement each other. Proposals for the existence of post-translational ER protein import in vivo are enhanced by supporting data from in vitro experiments (and vice versa), as SRP gene knockdowns do not prove the existence of a post-translational system (also see next paragraph).…”

Phosphorylation of the TATA-binding protein activates the spliced leader silencing pathway in Trypanosoma brucei

“…SLS is activated by persistent ER stress induced by pH changes or perturbation of protein translocation to the ER, which is achieved experimentally by depleting ER translocon proteins (16)(17)(18). Here, we found that silencing SEC63 increased the amounts of all the preinitiation transcription factors analyzed, suggesting that decreased SL RNA transcription leads to decreased turnover or increased synthesis of these proteins.…”

“…To examine whether other proteins involved in the transcription of SL RNA also change abundance and localization in trypanosomes exposed to ER stress, we stably expressed PTP-tagged TRF4, TFIIA2, or SNAP3 in T. brucei containing a tetracycline-inducible vector expressing a stem loop construct that promotes the degradation of SEC63 mRNA (18). Similar to SNAP2, the abundance of TRF4, TFIIA2, and SNAP3 was greater in cells exposed to tetracycline compared to those that were not (Fig.…”

Signal Recognition Particle: An Essential Protein-Targeting Machine

“…The best characterized thus far is the bacterial SecB/A system, which delivers bacterial secretory and outer-membrane proteins to the SecYEG complex and, through the ATPase cycles of SecA, drives the translocation of substrate proteins across the SecYEG translocon (1, 2). In yeast, the Sec62/63/71/72 system is a major pathway that mediates protein secretion (21, 22). Additional targeting pathways, including the Tat, Hsp70 and most recently the GET pathway, have been found (Fig.…”