Cotranslational Targeting and Posttranslational Translocation can Cooperate in Spc3 Topogenesis.

Secretory and membrane proteins follow either the signal recognition particle (SRP)-dependent cotranslational translocation pathway or the SRP-independent Sec62/Sec63-dependent posttranslational pathway for their translocation across the endoplasmic reticulum (ER). However, increasing evidence suggests that most proteins are cotranslationally targeted to the ER, suggesting mixed mechanisms. It remains unclear how these two pathways cooperate. Previous studies have shown that Spc3, a signal-anchored protein, requires SRP and Sec62 for its biogenesis. This study investigated the targeting and topogenesis of Spc3 and the step at which SRP and Sec62 act using in vivo and in vitro translocation assays and co-immunoprecipitation. Our data suggest that Spc3 reaches its final topology in two steps: it enters the ER lumen head-first and then inverts its orientation. The first step is partially dependent on SRP, although independent of the Sec62/Sec63 complex. The second step is mediated by the Sec62/Sec63 complex. These data suggest that SRP and Sec62 act on a distinct step in the topogenesis of Spc3.

Profiling of signal sequence characteristics and requirement of different translocation components.

The N-terminal signal sequence (SS) on proteins targeted to the endoplasmic reticulum (ER) is surprisingly diverse in hydrophobicity, in the number of preceding N-terminal residues (N-length), and in charged residues flanking the sequence. However, it remains unclear how these sequences despite their heterogeneity bind to the same site and open the Sec61 translocon. We assessed varying features of SSs and their efficiencies in initiating translocation across the ER by using 5-min radiolabeling in yeast. We found that while hydrophobic SSs with a short N-length efficiently initiated translocation in Sec62 mutant, Sec63 mutant and Sec72 deletion strains, most SSs showed varying degrees of translocation defect in these strains. In particular, Sec71 was required for internal hydrophobic SSs to efficiently initiate translocation. These results suggest that different combinations of Sec62, Sec63, Sec71 and Sec72 dynamically associate with the Sec61 translocon for the optimal binding of incoming SSs of broad characteristics and the initiation of protein translocation in vivo.

The Sec62-Sec63 translocon facilitates translocation of the C-terminus of membrane proteins.

The Sec62-Sec63 complex mediates post-translational translocation of a subset of primarily secretory proteins into the endoplasmic reticulum (ER) in yeast. Therefore, it has been thought that membrane proteins, which are mainly co-translationally targeted into the ER, are not handled by the Sec62-Sec63 translocon. By systematic analysis of single and multi-spanning membrane proteins with broad sequence context [with differing hydrophobicity, flanking charged residues and orientation of transmembrane (TM) segments], we show that mutations in the N-terminal cytosolic domain of yeast Sec62 impair its interaction with Sec63 and lead to defects in membrane insertion and translocation of the C-terminus of membrane proteins. These results suggest that there is an unappreciated function of the Sec62-Sec63 translocon in regulating topogenesis of membrane proteins in the eukaryotic cell.

Sec62 protein mediates membrane insertion and orientation of moderately hydrophobic signal anchor proteins in the endoplasmic reticulum (ER).

Nascent chains are known to be targeted to the endoplasmic reticulum membrane either by a signal recognition particle (SRP)-dependent co-translational or by an SRP-independent post-translational translocation route depending on signal sequences. Using a set of model and cellular proteins carrying an N-terminal signal anchor sequence of controlled hydrophobicity and yeast mutant strains defective in SRP or Sec62 function, the hydrophobicity-dependent targeting efficiency and targeting pathway preference were systematically evaluated. Our results suggest that an SRP-dependent co-translational and an SRP-independent post-translational translocation are not mutually exclusive for signal anchor proteins and that moderately hydrophobic ones require both SRP and Sec62 for proper targeting and translocation to the endoplasmic reticulum. Further, defect in Sec62 selectively reduced signal sequences inserted in an N(in)-C(out) (type II) membrane topology, implying an undiscovered role of Sec62 in regulating the orientation of the signal sequence in an early stage of translocation.