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. 2010 Aug 13;285(42):32671–32677. doi: 10.1074/jbc.M110.128256

FIGURE 1.

FIGURE 1.

Sequence alignment of Sss1p/Sec61γ homologs and the structure and function of Sss1p domain mutants. A, S. cerevisiae (Sc) Sss1p aligned with its homologs from Schizosaccharomyces pombe (Sp), Neurospora crassa (Ns), Mus musculus (Mm), Homo sapiens (Hs), Canis familaris (Cf), and Caenorhabditis elegans (Ce). Alignments were performed using CLUSTAL_X (16), with black, dark gray, and light gray shading representing 100%, 80%, and 60% amino acid conservation. The TM domain (2) is underlined, and the highly conserved glycine residues are indicated by arrows. B, diagram of the Sec61-translocon in the ER membrane as viewed from the back of the structure where the Sss1p TM domain is situated (see Ref. 1), illustrating the key features of the Sec61p-Sss1p interface. The long curved TM domain of Sss1p has contacts with Sec61p TM domains 1, 5, 6, and 10 (numbered, behind the Sss1p TM in this view). The cytosolic domain is orientated at ∼90° to its TM domain (direction of the arrow), having contact with the cytosolic loop between Sec61p TM 6 and 7 (6/7), and further toward the N terminus is in close proximity to Sec61p TM9 (9, behind the 6/7 loop in this view). C, structure of Sss1p domain mutants. Sss1p (8.9 kDa) and Ubc6p (28.3 kDa) are ER tail-anchored membrane proteins with TM sequences as indicated. The Sss1ΔCp (5.9 kDa) is truncated after residue Lys52 of Sss1p, and the Sss1Δ12p (7.5 kDa) is truncated after residue Ile68. USCp (29.6 kDa) consists of the cytosolic domain of Ubc6p (Met1–Ser232) fused to the C-terminal region of Sss1p (Ala53–Val80). NSUp (9.5 kDa) consists of the cytosolic domain of Sss1p (Met1–Lys52) fused to the TM of Ubc6p (Met233–Met249) with Sss1p residues Lys69–Val80 at the C terminus. D, functional analysis of Sss1p domain mutants. Vector and plasmids encoding SSS1, the Sss1ΔC, USC, and NSU mutants were transformed into the SSS1 plasmid shuffle strain BWY530. All transformant strains grew normally after 3 days on minimal medium with selection for the SSS1, URA3 plasmid (FKp53), and the HIS3 (pRS313)-transformed plasmids. After 3 days on 5-FOA containing minimal medium, the vector-only strain was unable to grow due to the lethality of the Δsss1 mutation upon FKp53 counterselection, whereas the wild-type plasmid (SSS1) transformant grew by providing Sss1p function. However, the Sss1ΔCp-, NSUp-, and USCp-encoding plasmids were unable to support growth, indicating the lethality of deletion and partial substitution of the Sss1p TM domain and the substitution of the Sss1p cytosolic domain, respectively.