Expression, purification, and characterization of Sss1p, an essential component of the yeast Sec61p protein translocation complex.
Sss1p, a 8.9-kDa membrane protein, is an essential component of the protein translocation complex involved in the transport of secretory proteins across the Saccharomyces cerevisiae endoplasmic reticulum membrane. In order to determine the high resolution structure of Sss1p by NMR, we have undertaken its overexpression and purification. We first inserted the yeast SSS1 gene into the pGEX-2T plasmid expression vector. Sss1p was expressed as fusions with Schistosoma japonica glutathione S-transferase (GST-Sss1p) in MC1061 Escherichia coli cells. Maximum yield of GST-Sss1p was obtained from cells harvested 2 h after induction at 37 degreesC in Luria broth medium. GST-Sss1p was found associated predominantly with the membrane pool and was readily extracted with Triton X-100. Detergent-solubilized GST-Sss1p was isolated by adsorption on glutathione-agarose beads. Sss1p was released from its GST carrier by cleavage with thrombin and its recovery was maximized by addition of dodecyl maltoside. Desorbed Sss1p was loaded on a high-performance liquid chromatography hydroxyapatite column equilibrated in phosphate buffer supplemented with dodecyl maltoside and the fractions containing Sss1p were subsequently purified to homogeneity by reverse-phase chromatography on a C4 column. The entire purification protocol can be completed in 5-6 h and yields about 0.4 mg of Sss1p per gram of transformed cells. CD and preliminary 1H NMR experiments show that purified Sss1p solubilized in SDS micelles is very stable and adopts a helical secondary structure.
Beswick
V
V
Département de Biologie Cellulaire et Moléculaire, Section de Biophysique des Protéines et des Membranes, URA CNRS 2096,
Brodsky
J L
JL
Képès
F
F
Neumann
J M
JM
Sanson
A
A
Garrigos
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005982:Glutathione Transferase / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000069816:SEC Translocation Channels; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins
D017931:DNA Primers; D005656:Fungal Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; C499383:SEC61 protein, S cerevisiae; C084005:SSS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D005982:Glutathione Transferase