Detection of transient in vivo interactions between substrate and transporter during protein translocation into the endoplasmic reticulum

Abstract

The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-alpha-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-alpha-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.

Figure 1

The split-Ub technique and its application to the analysis of protein translocation. (A) N_ub and C_ub are linked to the interacting proteins X and Y. The X–Y complex brings N_ub and C_ub into close proximity. N_ub and C_ub reconstitute a quasi-native Ub moiety, which is cleaved by the UBPs, yielding the free reporter R (Johnsson and Varshavsky, 1994a). (B) Using split-Ub to monitor the proximity between a secretory protein and a component of the translocation machinery. A signal sequence-bearing C_ub fusion (SS-Y-C_ub-R) and a N_ub fusion (N_ub-X) are coexpressed in a cell. Pathway 1: when N_ub is linked to a protein not involved in the targeting for translocation, the uncleaved (except for the signal sequence SS) Y-C_ub-R enters the ER. Pathway 2: when N_ub is linked to a protein involved in the targeting for translocation the signal sequence of the SS-Y-C_ub-R brings N_ub and C_ub into close contact. As a result, some of the SS-Y-C_ub-R and N_ub-X molecules interact to form the quasi-native Ub, yielding the free reporter R in the cytosol.

Figure 2

N_ub and C_ub test fusions. (A) N_ub (residues 1–36 of Ub) was fused to the N terminus of either a transmembrane protein (constructs 1–6) or a cytosolic protein (construct 7). The N termini of all proteins are located in the cytosol. The orientations and the numbers of membrane-spanning regions (shaded boxes) were derived from the published studies of these proteins, except for Ste14p, for which the exact number of the domains and the localization of the C terminus are not yet known. The N_ub fusions 1–5 are located in the membrane of the ER; the N_ub fusion 6 resides in the membrane of the early Golgi. The N_ub fusion 2 is a Sec62p derivative lacking the C-terminal 60 residues. The N_ub fusion 7 contains the full-length triosephosphate isomerase (N_ub-Tpi1p). (B) C_ub fusions. The C_ub fusions 8 and 9 contain the signal sequence of the prepro-α-factor (shaded boxes), followed by either 37 (construct 8) or 65 residues (construct 9) of the mature α-factor sequence (striped boxes) and a 7-residue linker sequence (not shown). C_ub fusions 10–13 contain the signal sequence of the Suc2p invertase (dark boxes) followed by 23 (construct 10), 33 (construct 11), 59 (construct 12), or 518 residues (construct 13) of the mature sequence of invertase (open boxes) and a 7-residue linker sequence (not shown). The C_ub fusion 14 contains the complete sequence of S. cerevisiae triosephosphate isomerase (Tpi1p) followed by a 17-residue linker peptide and C_ub. The C_ub fusions 15 and 16 are the signal sequence-lacking counterparts of the fusions 10 and 12. C_ub is always followed by a reporter protein. The reporter is DHFR-ha or Ura3p for the C_ub fusions 8–13, and DHFR-ha for the C_ub fusions 14, 15, and 16.

Figure 3

Sec62p is close to the signal sequence of the α-factor precursor. (A) S. cerevisiae cells expressing Mfα₃₇-C_ub-Dha (construct 8; Figure 2) were labeled for 5 min with ³⁵S-methionine. The extracted proteins were immunoprecipitated with anti-ha antibody, followed by a mock treatment (lanes a, c, e, and g) or the treatment with EndoH (lanes b, d, f, and h), and SDS-PAGE. The results with cells coexpressing N_ug- or N_ub-Sec62p are shown in lanes c and d and g and h, respectively. The analysis was performed with N_ug-Sec62p in the S. cerevisiae mutant RSY529 carrying a ts allele of SEC62 (lanes a–d) or with N_ub-Sec62p in the wild-type yeast (lanes e–h). Number 8 (following the numbering of the constructs in Figure 2) on the right indicates the positions of uncleaved Mfα₃₇-C_ub-Dha and its glycosylated forms. An asterisk denotes an unrelated yeast protein that cross-reacts with the anti-ha antibody. (B) N_ub-Sec62p encodes a functionally active protein. RSY529 cells carrying an empty plasmid (a), Sec62p (b), N_ub-Sec62p (c), Sec62(ΔC60)Dha (d), or N_ub-Sec62(ΔC60)Dha (e) were spotted on minimal media and grown for 2 d at 30°C (semipermissive temperature for unmodified RSY529).

Figure 4

The in vivo proximity between Sec62p and Mfα₃₇-C_ub-Dha is transient and specific. (A) Immunoblot analysis of extracts of S. cerevisiae coexpressing the Mfα₃₇-C_ub-Dha (construct 8; Figure 2) and one of the following constructs: N_ub-Sec62p, integrated (lane a) or plasmid-borne (lane b); N_ub-Bos1p (lane c); N_ub-Ste14p (lane d); N_ub-Sed5p(lane e); and N_ub-Tpi1p (lane f). (B) Immunoblot analysis of extracts of S. cerevisiae expressing the Tpi1p-C_ub-Dha fusion (construct 14; Figure 2) alone (lane a) or together with one of the following constructs: N_ub-Sec62p, either integrated (lane b) or plasmid-borne (lane c); N_ub-Bos1p (lane d); N_ub-Ste14p (lane e); N_ub-Sed5p(lane f); and N_ub-Tpi1p (lane g). Number 14 on the left indicates the position of uncleaved Tpi1p-C_ub-Dha. (C) S. cerevisiae cells expressing Mfα₃₇-C_ub-Dha (construct 8; Figure 2) together with either the vector (lane a), N_ub-Bos1p (lane b), or N_ub-Sec62p (lane c) were labeled for 5 min with ³⁵S-methionine. The extracted proteins were immunoprecipitated with anti-ha antibody and analyzed by SDS-PAGE. (D) Quantitation of the pulse-labeling experiment (C) using Phosphor-Imager. The extent of Dha release in the presence of N_ub-Sec62p was arbitrarily set at 100. The averages of three experiments are shown. Lanes a, b, and c are the same as in panel C. (E) S. cerevisiae cells expressing Mfα₃₇-C_ub-Dha together with N_ub-Sec62p were labeled for 5 min with ³⁵S-methionine and chased for 5 and 15 min, followed by extraction of proteins, immunoprecipitation with anti-ha antibody, and SDS-PAGE.

Figure 5

The nature of the signal sequence and its distance from C_ub determine the extent of cleavage of C_ub-R in the presence of N_ub-Sec62p. (A) S. cerevisiae expressing Mfα₃₇-C_ub-Dha (construct 8; Figure 2) (lanes a and b), Mfα₆₅-C_ub-Dha (construct 9) (lanes c and d), Suc2₂₃-C_ub-Dha (construct 10) (lanes e and f), Suc2₃₃-C_ub-Dha (construct 11) (lanes g and h), Suc2₅₉-C_ub-Dha (construct 12) (lanes i and j) and Suc2₅₁₈-C_ub-Dha (construct 13) (lanes k and l) were labeled with ³⁵S-methionine for 5 min. The extracted proteins were either mock-treated (lanes a, c, e, g, i, and k) or treated with EndoH (lanes b, d, f, h, j, and l), followed by immunoprecipitation with anti-ha antibody and SDS-PAGE. (B) Same as panel A but the cells also contained N_ub-Sec62p in addition to the Cub-fusions Mfα₃₇-C_ub-Dha, Mfα₆₅-C_ub-Dha, Suc2₂₃-C_ub-Dha, Suc2₃₃-C_ub-Dha, Suc2₅₉-C_ub-Dha, Suc2₅₁₈-C_ub-Dha (lanes a–f). The analysis was carried out by immunoblotting whole- cell extracts with the anti-ha antibody. (C) S. cerevisiae cells expressing Suc2₂₃-C_ub-Dha (construct 10; Figure 2) (lanes a–c) and Suc2₅₉-C_ub-Dha (construct 12; Figure 2) (lanes d–f) together with either N_ub-Sec62p (lanes b and e), N_ub-Bos1p (lanes c and f) or the vector (lanes a and d) were labeled for 5 min with ³⁵S-methionine. Whole-cell extracts were immunoprecipitated with anti-ha antibody, followed by SDS-PAGE and autoradiography. (D) S. cerevisiae cells expressing ΔSuc2₂₃-C_ub-Dha (construct 15; Figure 2) or ΔSuc2₅₉-C_ub-Dha (construct 16; Figure 2) together with either the vector (first six lanes) or N_ub-Sec62p (last six lanes) were labeled for 5 min with ³⁵S-methionine and chased for 10 and 30 min, followed by extraction, immunoprecipitation with anti-ha antibody, and SDS-PAGE. Numbers 15 and 16 indicate the positions of the corresponding (uncleaved) C_ub fusions.

Figure 6

Sec61p, but not a mutant of Sec62p, are close to the nascent chain of a translocated protein. (A) These assays employed S. cerevisiae expressing Mfα₃₇-C_ub-Dha (construct 8, Figure 2) and one of the following N_ub fusions (Figure 2): N_ub-Sec62p, either integrated (lane a) or plasmid borne (lane b); N_ub-Sec62(ΔC60)Dha (lane c); and N_ub-Sec61p (lane d). Whole-cell extracts from these strains were subjected to immunoblot analysis with anti-ha antibody. (B) Same as panel A but the same N_ub fusions were coexpressed with Tpi1-C_ub-Dha (construct 14; Figure 2). Numbers 2 and 14 indicate the positions of the corresponding (uncleaved) fusions. (C) Lane a: S. cerevisiae expressing Suc2₂₃-C_ub-Dha (construct 10; Figure 2) together with either N_ub-Sec61p, N_ua-Sec61p, N_ug-Sec61p, N_ub-Sec62p, N_ua-Sec62p, or N_ug-Sec62p; lane b: same as lane a but cells expressed Mfα₃₇-C_ub-Dha (construct 8; Figure 2) instead of Suc2₂₃-C_ub-Dha. (D) S. cerevisiae cells expressing Mfα₃₇-C_ub-Dha together with N_ub-Sec61p (a and b) or N_ub-Sec62p (e and f), and cells expressing Suc2₂₃-C_ub-Dha together with N_ub-Sec61p (c and d) or N_ub-Sec62p (g and h) were labeled for 5 min with ³⁵S-methionine. Whole-cell extracts were immunoprecipitated with anti-ha antibody, followed by SDS-PAGE, and quantitation of the cleaved and uncleaved C_ub fusions using PhosphorImager. Shown are the relative amounts of the cleaved (white bars: a, c, e, and g) and uncleaved (black bars: b, d, f, and h) C_ub fusions. The sum of a cleaved and uncleaved fusion was set at 100 in each of the three independent experiments. SDs are also indicated.

Figure 7

The use of a metabolic marker to assess the proximity between a component of the translocation machinery and a translocated protein. S. cerevisiae expressing the C_ub fusions 8–12 (Figure 2) that contained Ura3p instead of Dha (see the main text) were transformed with the vector (A) or plasmids expressing N_ub-Sec62p (B), N_ub-Sec61p (C), N_ub-Sec62(ΔC60)Dha (D), and N_ub-Bos1p (E). Cells were grown in a liquid uracil-containing SD medium, and ∼10⁵, 10³, and 10² cells were spotted onto uracil-lacking SD medium. Plates were examined after 18 h at 30°C.