Modes of substrate discrimination by the Sec61 translocon in Saccharomyces cerevisiae

Abstract

In eukaryotes, all secretory proteins contain a degenerate, hydrophobic stretch of sequence that targets them from the cytosol to the endoplasmic reticulum. This delivery step to the ER can occur in a translation-arrested or a fully translated nascent peptide state, which is determined in part by the hydrophobicity of the signal sequence. Hydrophobic signal sequences can act also as transmembrane segments, and are embedded into the ER membrane, while weakly hydrophobic signal sequences are generally cleaved off upon translocation into the ER. Irrespective of their targeting route to the ER, their routes merge at the Sec61 translocon. This pore-forming Sec61p facilitates a lateral exit of putative transmembrane segments through its proposed lateral gate (TM2a and TM7) or ER translocation of secretory proteins. As well as the gate, it contains a central constriction ring and a luminal plug domain. Sec61p has been shown to set the hydrophobicity threshold for incoming signal sequences and to discriminate authentic signal sequences over those that are not. The main aim of this study was to examine whether or not Sec61p (more specifically, different domains of Sec61p) recognizes different types of signal sequences distinctly. Firstly, in order to find substrates with signal sequences that are distinctly recognized by Sec61p, we 1) systematically defined the threshold N-terminal length and 2) the threshold signal sequence hydrophobicity for efficient ER translocation across the Sec61 translocon taking CPY as our model protein. We found that a short N-tail length is required for translocation of CPY with weakly hydrophobic signal sequences, while those with long N-tail length were only translocated when the signal sequence was hydrophobic. Using these two types of signal sequences that may be differentially recognized by the Sec61 translocon, their translocation were tested in Sec61 mutants of the plug domain and the lateral gate. We found that signal sequences with short N-tails and weakly hydrophobic signal sequences are more sensitive to changes in plug domain residues and mutations in TM7 of the lateral gate than those with long N-tails and hydrophobic signal sequences, thereby demonstrating differential substrate recognition by Sec61.