Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

Abstract

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.

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

“…No N-terminal signal sequence exists in this protein, suggesting that the first transmembrane domain may function in its targeting. The predicted topology of the T. brucei protein based on the Kyte-Doolittle method (40) agrees well with that of its yeast homologue (14). The yeast proteins possess two potential glycosylation sites in the luminal region upstream to the predicted transmembrane domain.…”

“…Trypanosomes Possess a SEC71 Homologue-To study the role of the protein translocation SRP-independent pathway in T. brucei, the genome was first searched for a homologue to SEC71, which participates in protein translocation across the ER in yeast (10,14). A SEC71 homologue was identified only after searching for this protein with a profile that was generated based on multiple alignments of this protein in yeast and other fungi (see Fig.…”

“…The SEC complex is composed of Sec63p, Sec62p, Sec71p, and Sec72p. The first two genes, SEC62 and SEC63, are essential for growth, whereas SEC71 and SEC72 are not (14). Mutations in SEC62, SEC71, and SEC72 predominantly affect post-translationally translocated precursors, suggesting that these gene products may be specific for this pathway (10).…”

Suppression of a sec63 mutation identifies a novel component of the yeast endoplasmic reticulum translocation apparatus.

“…The high copy suppression of sec63-101 by HSS1 is also consistent with a functional interaction between HSSlp and Sec63p. The genetic predictions for HSSlp-Sec63p interaction were confirmed by the identity of HSSlp with gp3l.5 (Sec66p), a protein that is found in an ER membrane-bound complex (Sec complex) with Sec6lp, Sec62p, Sec63p, and a 23-kDa protein (Deshaies et al, 1991;Feldheim et al, 1993).…”

“…When HSS1-myc was expressed from its own promoter in a Ahssl strain (PSY396), a mostly perinuclear myc staining pattern was seen (Figure 6) similar to that seen for ER proteins such as Kar2p and Sec63p (Rose et al, 1989;Feldheim et al, 1992 Sec6lp, Sec62p, and Sec63p (Deshaies et al, 1991). SEC66, the gene encoding gp31.5, has been cloned (Feldheim et al, 1993), and comparison of HSS1 and SEC66 sequences indicates that they are identical.…”