Probing the molecular environment of membrane proteins in vivo.
The split-Ubiquitin (split-Ub) technique was used to map the molecular environment of a membrane protein in vivo. Cub, the C-terminal half of Ub, was attached to Sec63p, and Nub, the N-terminal half of Ub, was attached to a selection of differently localized proteins of the yeast Saccharomyces cerevisiae. The efficiency of the Nub and Cub reassembly to the quasi-native Ub reflects the proximity between Sec63-Cub and the Nub-labeled proteins. By using a modified Ura3p as the reporter that is released from Cub, the local concentration between Sec63-Cub-RUra3p and the different Nub-constructs could be translated into the growth rate of yeast cells on media lacking uracil. We show that Sec63p interacts with Sec62p and Sec61p in vivo. Ssh1p is more distant to Sec63p than its close sequence homologue Sec61p. Employing Nub- and Cub-labeled versions of Ste14p, an enzyme of the protein isoprenylation pathway, we conclude that Ste14p is a membrane protein of the ER. Using Sec63p as a reference, a gradient of local concentrations of different t- and v-SNARES could be visualized in the living cell. The RUra3p reporter should further allow the selection of new binding partners of Sec63p and the selection of molecules or cellular conditions that interfere with the binding between Sec63p and one of its known partners.
Wittke
S
S
Max-Delbrück-Laboratorium, D-50829 Köln, Germany.
Lewke
N
N
Müller
S
S
Johnsson
N
N
D001483:Base Sequence; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D011496:Protein Methyltransferases / Q000235:genetics / Q000378:metabolism; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins
C068037:BOS1 protein, S cerevisiae; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; C074213:SEC62 protein, S cerevisiae; C074215:SEC63 protein, S cerevisiae; C084006:SSO1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C077554:Sed5 protein, S cerevisiae; D014452:Ubiquitins; C107706:VAM3 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase