Signal Peptide Features Determining the Substrate Specificities of Targeting and Translocation Components in Human ER Protein Import

Abstract

In human cells, approximately 30% of all polypeptides enter the secretory pathway at the level of the endoplasmic reticulum (ER). This process involves cleavable amino-terminal signal peptides (SPs) or more or less amino-terminal transmembrane helices (TMHs), which serve as targeting determinants, at the level of the precursor polypeptides and a multitude of cytosolic and ER proteins, which facilitate their ER import. Alone or in combination SPs and TMHs guarantee the initial ER targeting as well as the subsequent membrane integration or translocation. Cytosolic SRP and SR, its receptor in the ER membrane, mediate cotranslational targeting of most nascent precursor polypeptide chains to the polypeptide-conducting Sec61 complex in the ER membrane. Alternatively, fully-synthesized precursor polypeptides and certain nascent precursor polypeptides are targeted to the ER membrane by either the PEX-, SND-, or TRC-pathway. Although these targeting pathways may have overlapping functions, the question arises how relevant this is under cellular conditions and which features of SPs and precursor polypeptides determine preference for a certain pathway. Irrespective of their targeting pathway(s), most precursor polypeptides are integrated into or translocated across the ER membrane via the Sec61 channel. For some precursor polypeptides specific Sec61 interaction partners have to support the gating of the channel to the open state, again raising the question why and when this is the case. Recent progress shed light on the client spectrum and specificities of some auxiliary components, including Sec62/Sec63, TRAM1 protein, and TRAP. To address the question which precursors use a certain pathway or component in intact human cells, i.e., under conditions of fast translation rates and molecular crowding, in the presence of competing precursors, different targeting organelles, and relevant stoichiometries of the involved components, siRNA-mediated depletion of single targeting or transport components in HeLa cells was combined with label-free quantitative proteomics and differential protein abundance analysis. Here, we present a summary of the experimental approach as well as the resulting differential protein abundance analyses and discuss their mechanistic implications in light of the available structural data.

Molecular view of ER membrane remodeling by the Sec61/TRAP translocon

“…SRP recognition of secretory proteins is based upon interaction with a nascent N-terminal cleavable hydrophobic signal peptide or transmembrane segment. The large sequence diversity of targeting peptides suggests that their targeting and insertion mechanisms may vary (Lang et al 2022); (Liaci and Förster 2021)…”

“…SRP recognition of secretory proteins is based upon interaction with a nascent N-terminal cleavable hydrophobic signal peptide or transmembrane segment. The large sequence diversity of targeting peptides suggests that their targeting and insertion mechanisms may vary (Lang et al 2022); (Liaci and Förster 2021) The evolutionarily conserved heterotrimeric Sec61 channel is responsible for ER translocation of nascent polypeptides and is alone sufficient for translocation of nascent polypeptides across the ER membrane (Görlich and Rapoport 1993). However, a subset of secretory proteins contain signal peptides that are inefficient in engaging with Sec61 and cannot be translocated by Sec61 alone (Fons et al 2003); (Hegde et al 1998).…”

The endoplasmic reticulum membrane protein Sec62 as potential therapeutic target in SEC62 overexpressing tumors

“…These findings raised the question of how SEC62 overexpression causes increased stress tolerance and migratory and invasive potential of the respective cancer cells: 1) On first sight, the role of Sec62 in ER protein import may appear to be an unlikely candidate since the protein typically cooperates with Sec63 and simultaneous SEC63 overexpression was not observed in SEC62 overexpressing tumor cells (Greiner et al, 2011a). However, in our opinion it cannot entirely be dismissed since in some cases Sec62 was found to play a role in ER protein import without the involvement of Sec63 (Haßdenteufel et al, 2018;Lang et al, 2022). In this context it is tempting to speculate that the biogenesis of ADAM metalloproteinases, which are involved in proteolytic cleavage of extracellular matrix and cell adhesion proteins, as well as cadherins that play a direct role in cell adhesion, is limited by standard Sec62 levels.…”

Structural insights into TRAP association with ribosome-Sec61 complex, and translocon inhibition by a CADA derivative

“…The structural information on TRAP subunit assembly and their interactions with the ribosome-Sec61 translocon complex would facilitate a better understanding of the roles that TRAP play in protein translocation. Interestingly, the TRAP complex is present in our eEF2-bound non-translating ribosome-Sec61 translocon complex; the structure, therefore, implies that TRAP is an integral part of the Sec61 translocon network (19,23).…”

“…Other accessory translocation proteins, such as the heterotetrametric TRanslocoAssociated Protein (TRAP) complex have been identified for which different functions have been proposed (13,14). TRAP, composed of α, β, γ, and δ (ssr1 -ssr4) subunits, is thought to be facilitating the translocation of substrates with weak (low hydrophobicity) or proline and glycine-rich SPs, by ensuring the opened conformation of Sec61 (15)(16)(17)(18)(19). Furthermore, TRAP might interfere with the final topology of the protein nascent chain (17) and subsequently coordinate with OST for the N-linked glycosylation, which suggests a role of TRAP in the biogenesis of glycoproteins (20,21).…”