YEAST PROTEIN TRANSLOCATION COMPLEX - ISOLATION OF 2 GENES SEB1 AND SEB2 ENCODING PROTEINS HOMOLOGOUS TO THE SEC61-BETA SUBUNIT

A yeast gene (cDNA clone) was isolated in a screen for suppressors of secretion-defective sec15-1 mutation. This gene encodes a protein homo logous to the beta subunit of the mammalian Sec61 protein complex func tioning in protein translocation into the endoplasmic reticulum (ER). The predicted protein, Seb1p, consists of 82 amino acids and contains one potential membrane-spanning region at the C-terminus but no N-term inal signal sequence. Seb1p shows 30% identity to the mammalian Sec61 beta subunit and 34% identity to the Arabidopsis thaliana Sec61 beta s ubunit. Overexpression of SEB1 from a multicopy plasmid suppressed the temperature sensitivity of sec61-2 and sec61-3 mutants. Immunofluores cence and immunoelectron microscopy indicated that Seb1p resides in th e ER membranes with the hydrophilic N-terminus exposed to the cytoplas m. The in vitro translated Seb1p was post-translationally inserted int o microsomal membranes. As the chromosomal disruption of the SEB1 gene was not lethal, potential homologous genes were screened by heterolog ous hybridization. The SEB1 homologue thus isolated, SEB2, encodes a p rotein 53% identical to Seb1p. Disruption of the chromosomal SEB2 was not lethal whereas the double disruption of SEB1 and SEB2 resulted in a temperature-sensitive phenotype. This study further emphasizes the e volutionary conservation of the ER protein translocation apparatus and provides novel genetic tools for its functional analysis, The sequenc es of SEB1 and SEB2 have been deposited in the EMBL database under Acc ession Numbers 247789 and Z50012, respectively. The sequence of the Se b1 protein is identical to that of Sbh1p independently identified by P anzner et al. (Cell 81, 561-570. 1995) using a biochemical approach.