Probing the molecular environment of membrane proteins in vivo - PubMed (original) (raw)

Probing the molecular environment of membrane proteins in vivo

S Wittke et al. Mol Biol Cell. 1999 Aug.

Free PMC article

Abstract

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.

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Figures

Figure 1

Figure 1

The split-Ubiquitin technique and its application to the analysis of membrane proteins using a metabolic marker. Cub-RUra3p was linked to the C terminus of Sec63p, and Nub was linked to the N terminus of the membrane protein X. Pathway 1: Nub is coupled to a protein that binds to Sec63p. The complex brings Nub and Cub into close proximity. Nub and Cub reconstitute the quasi-native Ub that is cleaved by the Ub-specific proteases to release RUra3p from Cub. The cleaved RUra3p is targeted for rapid destruction by the enzymes of the N-end rule (3) to yield cells that are uracil auxotrophs and 5-FOA resistant. Pathway 2: Nub is linked to a protein that does not bind to Sec63p. The two fusion proteins do not improve the reconstitution of Nub and Cub into the quasi-native Ub. Thus, RUra3p stays linked to Sec63-Cub, and the cells are uracil prototrophs and 5-FOA sensitive.

Figure 2

Figure 2

Nub and Cub fusions. (A) Nub (residues 1–36 of Ub) was fused to the N terminus of either a transmembrane protein (constructs 1–11) or a cytosolic protein (constructs 12–13). The N termini of all proteins are located in the cytosol. The orientation and the numbers of the membrane-spanning domains were obtained from published studies. The orientation of the N and the C terminus of Ste14p and its subcellular localization was a subject of this study. The Nub-attached proteins of constructs 1–5 are localized in the ER (Deshaies and Schekman, 1990; Shim et al., 1991; Finke et al., 1996; Wilkinson et al., 1996; Ballensiefen et al., 1998). The localization of the Nub-attached protein of construct 6 was a subject of this study. The Nub-attached protein of construct 7 resides in the early Golgi and of construct 8 in the late Golgi/plasma membrane (Protopopov et al., 1993; Banfield et al., 1994). The Nub-attached protein of construct 9 was shown to be in the plasma membrane (Aalto et al., 1993). The Nub-attached protein of construct 10 was found in the vacuole, and the Nub-attached protein of construct 11 was found in the outer membrane of the mitochondrion (Kiebler et al., 1993; Darsow et al., 1997; Wada et al., 1997; Srivastava and Jones, 1998). (B) Cub (residues 35–76 of Ub) was linked to the C terminus of a transmembrane protein and extended at its own C terminus by a reporter protein. The C termini of all proteins are localized in the cytosol. The information on the orientation of the N- and C-termini, the numbers of the membrane-spanning domains, and the localization of the unmodified proteins were obtained from published studies except for construct 15, where the number of membrane-spanning domains is still tentative. The Cub-attached protein of construct 14 is localized in the ER, that of construct 16 is found in the plasma membrane, and that of construct 17 is localized in the outer membrane of the mitochondrion (Jund et al., 1988; Feldheim et al., 1992; Moczko et al., 1997). The reporter (R) is RUra3p for the constructs 15–17 and RUra3p or DHFRha (Dha) for construct 14.

Figure 3

Figure 3

Split-Ub monitors the interaction between Sec63p and Sec62p in vivo. (A) Immunoblot analysis of cells expressing Sec63-Cub-Dha together with an empty plasmid (lane a) or together with Nub-, Nua-, or Nug-Sec62p (lanes b, c, and d, respectively) or Nub-, Nua-, or Nug-Bos1p (lanes e, f, and g, respectively). The nitrocellulose membrane was probed with the anti-ha antibody that recognizes the uncleaved Cub fusion and the cleaved Dha. (B) Growth assay of the interaction between Sec63p and Sec62p based on split-Ub and a short-lived Ura3p (RUra3p) as a reporter. Sec63CRUp-containing cells bearing either the UBR1 gene or a UBR1 deletion were transformed with an empty plasmid or Nub-, Nua-, or Nug-Sec62p. Cells were pregrown in selective media containing uracil. Cells (103 or 102) were spotted on selective plates lacking uracil and also lacking leucine and tryptophan to select for the presence of the Cub- and Nub-constructs.

Figure 4

Figure 4

The measured proximity between Sec62p and Sec63p is due to both proteins being in one complex. (A) Cells bearing Sec63CRUp and Nug-Sec62p were transformed with a plasmid containing either Sec62p, Sec62Dha, Ste14Dha, Tpi1ha, or an empty plasmid, all under the control of the PGAL1-promoter (lanes a–e). Approximately 105, 104, 103, and 102 cells were spotted on selective media lacking uracil and containing either glucose to repress or galactose to induce the PGAL1 promoter. (B) S. cerevisiae cells (104) were plated as described in panel A on selective media containing galactose and lacking uracil, and colonies were counted after 4 d. The average of seven independent experiments is shown. Approximately 800 colonies were recovered upon overexpression of Sec62p. This number was arbitrarily set as 100. (C) Overexpression of the ha epitope-bearing proteins was confirmed by immunoblot analysis of extracts of S. cerevisiae cells coexpressing Sec63CRUp, Nug-Sec62p, and the following constructs: Tpi1ha (lanes a and f), Ste14Dha (lanes b and g), Sec62Dha (lanes c and h), Sec62p (lanes d and i), and empty vector (lanes e and j). Cells were grown in glucose (lanes a–e) to repress and grown in galactose (lanes f–j) to induce the expression of the proteins.

Figure 5

Figure 5

Split Ub measures the proximity between Sec63p and membrane-associated proteins in vivo. Sec63CRUp containing cells expressing Nub, Nua, and Nug constructs of Sec62p (A), Sec61p (B), Ssh1p (C), Bos1p (D), Ste14p (E), Sed5p (F), Sso1p (G), Snc1p (H), Tom22p(I), Vam3p (J), Tpi1p (K), and Guk1p (L) were spotted (105 and 103 cells) on selective media lacking uracil (A–M) and leucine and histidine (A and D) or leucine and tryptophan (B, C, and E–M) to select for the presence of the Cub and Nub constructs. (M) Sec63CRUp-containing cells bearing either the empty plasmid, Nub-, Nua-, -Nug-Sec22p or Nub-, Nua-, Nug-Sec61p were spotted (105, 104, 103 cells) on plates lacking uracil. Cells were grown for 4 d.

Figure 6

Figure 6

(A) Nub and Cub constructs of Ste14p are functional. Nub-Ste14p and Ste14CRUp were expressed in cells containing a STE14 deletion and mated with an appropriate tester strain of the opposite mating type. The mated cells were patched on media selecting for the formation of diploids. (B) Ste14p is located between Bos1p and Sed5p. Sec63CRUp containing cells expressing Nvi-Sec62p (a),-Ssh1p (b),-Bos1p (c),-Ste14p (d),-Sed5p (e),-Sso1p (f), and -Snc1p (g) were spotted (105, 104, 103, and 102 cells) on SD-ura plates that also lacked leucine and tryptophan to select for the presence of the Cub and Nvi constructs. Cells were grown for 3 d. (C) Sec62p, Ssh1p, and Sec61p are equidistant to Ste14p. Ste14CRUp-containing cells expressing Nub, Nua, and Nug constructs of Sec62p (a), Ssh1p (b), Sec61p (c), Ste14p (d), Sed5p (e), and Sso1p (f) were spotted (105, 103, and 102 cells) on selective media lacking uracil, leucine, and tryptophan and containing 500 μM methionine to reduce the expression of Ste14CRUp. Cells were grown for 3 d.

Figure 7

Figure 7

Tom22p is close to Tom20p; Sso1p and Snc1p are close to Fur4p. (A) Tom20CRUp-containing S. cerevisiae cells expressing the Nub and Nua constructs of Tom22p (a), Sec62p (b), Sso1p (c), and Vam3p (d) were spotted (103 and 102 cells) on selective media lacking uracil. Cells were grown for 3 d. (B) Fur4CRUp containing S. cerevisiae cells expressing the Nub and Nua constructs of Sso1p (a), Snc1p (b), Sec62p (c), and Sed5p (d) were spotted (105 and 103 cells) on selective media lacking uracil. Cells were grown for 3 d. (C) Tom20CRUp-containing cells bearing the UBR1 gene or a UBR1 deletion were transformed with a plasmid harboring Nub-Tom22p or the empty vector pRS314. Cells (103 and 102) were spotted on selective media lacking uracil. Plates were incubated for 3 d.

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