McGill Cancer Centre, McGill University, Montréal, Québec H3G 1Y6, Canada
Author for correspondence (e-mail: annette{at}med.mcgill.ca)
Accepted September 13, 2001
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SUMMARY |
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Key words: Endoplasmic reticulum, Localization, Mannosidase, Retrieval, Protein interaction, Split-ubiquitin
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INTRODUCTION |
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Rer1p is a 22 kDa protein with four putative transmembrane domains (Boehm et al., 1994; Sato et al., 1995). It is structurally and functionally conserved among yeast, humans and plants (Füllekrug et al., 1997; Sato et al., 1999). Rer1p was initially shown to be involved in the recycling of the ER membrane protein Sec12p from an early Golgi compartment, mediated by the transmembrane domain of Sec12p (Boehm et al., 1994; Nishikawa and Nakano, 1993; Sato et al., 1996). Subsequently, Sec63p and Sec71p, two membrane proteins of different topologies, were also found to depend on Rer1p for retrieval from the Golgi to the ER (Sato et al., 1997). Similarly, a fusion protein consisting of Gas1p with an uncleavable signal sequence, attached to invertase was shown to depend upon Rer1p for its ER localization (Letourneur and Cosson, 1998). These studies indicate that Rer1p is required for localization of different types of ER membrane proteins and is likely to be part of a common recycling mechanism for retrieving membrane proteins from the Golgi to the ER.
Rer1p-dependent proteins mislocalize to the late Golgi in COPI temperature-sensitive yeast strains at the non-permissive temperature, indicating that the proteins are most likely recycled in COPI vesicles (Boehm et al., 1997; Sato et al., 1997). Rer1p itself is a dynamic protein that cycles between the Golgi and the ER but its steady state localization is the Golgi apparatus (Sato et al., 2001). It has been detected in COPII vesicles (Otte et al., 2001) and found to interact with components of the COPI complex (Sato et al., 2001).
The split-ubiquitin system used to monitor protein interactions in vivo was first described for cytosolic proteins by Johnsson and Varshavsky (Johnsson and Varshavsky, 1994). It was used to detect the interaction between signal sequences and a member of the ER translocation machinery (Dünnwald et al., 1999), to probe the environment of membrane proteins (Wittke et al., 1999), to study the interaction between transcriptional regulators (Laser et al., 2000; Rojo-Niersbach et al., 2000) and to determine the folding states of soluble proteins (Raquet et al., 2001). Ubiquitin is a 76 amino acid polypeptide that can be genetically split into N-terminal (NubI: amino acids 1 to 37) and C-terminal (Cub: amino acids 35 to 76) fragments. Co-expression of NubI and Cub as soluble cytosolic polypeptides leads to the assembly of the two fragments into a quasi-native structure that is a substrate for the cytosolic ubiquitin-specific proteases (UBPs). Several molecules have been fused to the C-terminal glycine residue of Cub to serve as reporters for the action of UBPs on the reconstituted ubiquitin molecule (Johnsson and Varshavsky, 1994; Rojo-Niersbach et al., 2000; Stagljar et al., 1998; Wittke et al., 1999). Mutation of isoleucine at position 13 in NubI to glycine (NubG) greatly decreases the affinity between NubG and Cub, preventing their interaction. However, when NubG and Cub are linked to two proteins that interact, these proteins promote the association between NubG and Cub, leading to the cleavage of the reporter molecule (Johnsson and Varshavsky, 1994).
The split-ubiquitin system has been applied to study membrane protein interactions that are difficult to identify using conventional yeast two-hybrid systems (Stagljar et al., 1998; Stagljar and te Heesen, 2000). In this modified split-ubiquitin system, Nub and Cub are attached to membrane-anchored proteins. A reporter group (PLV) consisting of Protein A (for detection of the protein with IgG), Lex A (DNA-binding protein) and the transcription activating domain of VP16 is linked to the C-terminal of Cub. Association of NubI and Cub-PLV leads to cleavage of PLV in the cytosol and activation of the ß-galactosidase reporter gene in the genome of L40 yeast cells. NubG does not spontaneously associate with Cub-PLV. However, when NubG and Cub-PLV are fused to two membrane proteins that interact, the proteins promote the association of NubG and Cub-PLV, resulting in cleavage of the PLV reporter molecule. When linked to membrane proteins, both Nub and Cub should be on the cytosolic side of the membrane where the UBPs are active (Stagljar et al., 1998; Stagljar and te Heesen, 2000).
In this work, we show that the transmembrane domain of Mns1p is responsible for its Rer1p-dependent ER localization. In order to elucidate the mechanism of Rer1p involvement in ER localization of Mns1p, we applied the split-ubiquitin system to determine whether these proteins interact. Using this method, we demonstrate that both Mns1p and Sec12p interact with Rer1p in vivo and that the C-terminal tail of Rer1p is on the cytosolic side of the membrane.
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MATERIALS AND METHODS |
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Construction of the Y23K97 chimera
The first 69 nucleotides of MNS1 were fused to KRE2, starting at nucleotide 289 using the splicing-by-overlap extension method. In a first round of PCR, the first 69 nucleotides of MNS1 were amplified using the sense primer C, which includes a PstI site (underlined), and the anti-sense primer D. KRE2 starting nucleotide 289 was amplified using the sense primer E and the anti-sense primer F with an XbaI site (underlined). The products of the first round of PCR were combined and reamplified using primers C and F leading to the formation of Y23K97. This construct was digested with PstI/XbaI and ligated into the multicopy plasmid pVT100U downstream of the ADH promoter to give pMM1.
Construction of pRS305(rer1-Cub-PLV) and integration at the RER1 locus of L40 cells
A truncated form of the RER1 gene lacking the N-terminal 219 nucleotides was amplified from genomic DNA using the sense primer G and anti-sense primer H. The resulting PCR product was used to replace wbp1 in pRS305(
wbp1-Cub-PLV) at XhoI sites (underlined in the primers). A silent mutation was introduced in the sense primer G, creating a unique PstI site (italic) used to linearize the vector. L40 cells, carrying the ß-galactosidase gene in their genome under the control of the VP16 promoter, were transformed with the linearized pRS305(
rer1-Cub-PLV) vector. Clones that correctly integrated the construct at the RER1 locus (MML40) were detected by PCR and western blotting.
Construction of pRS314(Nub-MNS1)
CUP1-NubG-MNS1 and CUP1-NubI-MNS1 were constructed using the splicing-by-overlap extension approach. In a first round of PCR, CUP1-NubG and CUP1-NubI were amplified with the sense primer I and the anti-sense primer J using pRS314(NubG-ALG5) and pRS314(NubI-Alg5) as templates, respectively. The MNS1 gene was amplified using the sense primer K and the anti-sense primer L that includes a PstI site (underlined). The products of the first round of PCR were combined and reamplified using primers I and L leading to the formation of CUP1-NubG-MNS1 and CUP1-NubI-MNS1. The CUP1-NubI-ALG5 cassette was removed from pRS314(NubI-ALG5) vector using NotI/PstI and replaced with either CUP1-NubG-MNS1 or CUP1-NubI-MNS1, leading to the formation of pRS314(NubG-MNS1) and pRS314(NubI-MNS1), respectively.
Construction of pRS314(Nub-SEC12)
CUP1-NubG and CUP1-NubI were amplified using the sense primer I and anti-sense primer M. The SEC12 gene was amplified using the sense primer N and the anti-sense primer O that includes a PstI site (underlined). The products of the first round of PCR were combined and reamplified using primers I and O, resulting in the formation of CUP1-NubG-SEC12 and CUP1-NubI-SEC12. The CUP1-NubI-ALG5 cassette was removed from pRS314(NubI-ALG5) vector using NotI/PstI and replaced with either CUP1-NubG-SEC12 or CUP1-NubI-SEC12, leading to the formation of pRS314(NubG-SEC12) and pRS314(NubI-SEC12), respectively.
Construction of pRS314(MNS1) and pRS314(CUP1)
The MNS1 gene and its promoter were excised from pYH4 using XbaI/XhoI and used to replace CUP1-NubI-ALG5 in the pRS314(NubI-Alg5) vector at the NotI/PstI sites, resulting in pRS314(MNS1). pRS314(CUP1) was derived from pRS314(NubI-ALG5) by removal of NubI-ALG5 with EcoRI and recircularization of the vector.
Immunofluorescence experiments
Indirect immunofluorescence was performed as described in Massaad et al. (Massaad et al., 1999). Briefly, yeast cells expressing Mns1p, Y23K97 or Kre2p on multi-copy plasmids pYH4, pVT100U and YEp352, respectively, were grown at 30°C in minimal medium to an OD600nm of 0.5-0.7. Cells fixed with paraformaldehyde were treated with Zymolyase 100T (Seikagaku Co.). The resulting spheroplasts were incubated in methanol followed by acetone. Mns1p, Y23K97 and Kre2p were detected with specific affinity-purified rabbit polyclonal antibodies (Burke et al., 1996; Lussier et al., 1995) at dilutions of 1/100 (Mns1p) and 1/50 (Y23K97 and Kre2p). These proteins were visualized using TRITC-conjugated affinity-purified goat anti-rabbit IgG (Jackson ImmunoResearch) at a dilution of 1/200. The 60 kDa subunit of the endogenous yeast vacuolar H+-ATPase was detected using mouse monoclonal antibody 13D11-B2 (Molecular Probes Inc.) at a dilution of 1/50, followed by CY2-conjugated affinity-purified goat anti-mouse IgG (Jackson ImmunoResearch) at a dilution of 1/200. Nuclei were visualized with DAPI. The cells were viewed with a Nikon Eclipse 800 epifluorescence microscope and photographed using TMY 400 film (Eastman Kodak).
Split-ubiquitin assay
The split-ubiquitin assay was performed according to (Stagljar et al., 1998). MML40 cells expressing endogenous levels of Rer1p-Cub-PLV were transformed with the different Nub plasmids. Individual colonies were streaked onto Whatman III filter paper and grown for 48 hours on selective medium containing 0.2 mM CuSO4 to induce the CUP1 promoter. Filters were immersed in liquid nitrogen for three minutes, allowed to warm up to room temperature then overlayed with 50 ml 1.5% agarose in 0.1 M NaPO4 (pH 7.0) containing 0.2 mM CuSO4 and 0.4 mg/ml 5-bromo-4-chloro-indolyl ß-D-galactopyranoside (X-gal; Gibco BRL), followed by incubation at 37°C for two hours.
Detection of PLV cleavage by western blot analysis
Cells were grown overnight at 30°C in 3 ml of selective medium containing 0.2 mM CuSO4 until OD600=1.0. Each culture was centrifuged five minutes at 2000xg, the pellet suspended in 50 µl 1.85 M NaOH per 3 OD600 of cells and incubated for 10 minutes on ice. An equal volume of 50% trichloroacetic acid was added, followed by incubation on ice for 10 minutes. Cells were centrifuged five minutes at 13000xg, the supernatant discarded and the pellet suspended in 50 µl SDS sample buffer containing 8 M urea and 20 µl 1 M Tris (Amersham Pharmacia) followed by boiling for five minutes. Twenty µl of extracts were resolved on 8% polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was probed with rabbit HRP-IgG (Cappel, ICN Pharmaceuticals) at a dilution of 1/500 and protein A was visualized using ECL (Amersham Pharmacia).
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RESULTS |
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To confirm that the X-Gal reaction was due to cleavage of the PLV reporter molecule, western blot analysis was done on MML40 cell extracts using IgG that recognizes protein A. In non-transformed MML40 cells, or in cells transformed with either vector control or Mns1p lacking the Nub portion, PLV is detected as the expected 74.5 kDa Rer1p-Cub-PLV (Fig. 5, lanes 1, 2 and 3). Expression of NubG-Mns1p and NubG-Sec12p leads to cleavage of PLV detected as a 49.5 kDa band (Fig. 5, lanes 5 and 7). Ost1p-NubG expressed in MML40 did not result in cleavage of PLV from Rer1p-Cub-PLV (Fig. 5, lane 9), whereas expression of NubG-Alg5p results in partial cleavage of the reporter group (Fig. 5, lane 11). PLV cleavage was detected with the NubI forms of all the fusion proteins (Fig. 5, lanes 4, 6, 8 and 10). Overall, there is a good correlation between the cleavage of the reporter molecule observed by western blot analysis and the X-gal assay.
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DISCUSSION |
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The transmembrane domain of Mns1p includes two serine and one threonine residue aligned on the same side of the -helix, as well as two tyrosine residues. To determine whether these polar residues play a role in the Rer1p-dependent ER localization of Mns1p, several types of transmembrane domain mutants were constructed. First, the two serine and one threonine residues were simultaneously changed to either three leucine or three alanine residues. Second, the two tyrosine residues were changed to either two leucine or two alanine residues, and third, the serine, threonine and tyrosine residues were all mutated to leucine. Indirect immunofluorescence indicated that the localization of Mns1p in wild-type cells was not affected by any of these mutations (data not shown). These data are in contrast to the results reported for Sec12p and Gas1p in which mutagenesis of polar residues affected their Rer1p-dependent ER localization (Letourneur and Cosson, 1998; Sato et al., 1996). The structural determinants of the Mns1p transmembrane domain responsible for its ER localization are therefore not defined.
This work demonstrates an interaction between Mns1p and Rer1p in vivo using a modified split-ubiquitin system. Being membrane proteins, Mns1p and Rer1p are poor candidates for the classical yeast two-hybrid system that depends on the ability of the tested proteins to localize to the nucleus (Fields and Song, 1989). The split-ubiquitin system is applicable to the study of membrane protein interactions and is very sensitive for the detection of transient interactions in vivo. This interaction may be either direct, through association between the transmembrane domain of Mns1p and one or more transmembrane domains of Rer1p, or indirect and mediated by another protein as part of a complex. Similarly, interaction between Sec12p and Rer1p was also observed using the split-ubiquitin system. This result is consistent with the observation that Sec12p partly depends on Rer1p for its ER localization (Nishikawa and Nakano, 1993; Sato et al., 1995; Sato et al., 1996). While the current work was in progress, a direct interaction between Rer1p and a chimeric protein consisting of the transmembrane domain of Sec12p fused to the cytoplasmic and luminal domains of the vacuolar protein Dap4p was demonstrated by crosslinking and co-immunoprecipitation (Sato et al., 2001). It is likely therefore that a similar direct interaction exists between Mns1p and Rer1p. In contrast, Ost1p, a subunit of the ER oligosaccharyltransferase complex (Silberstein et al., 1995) did not show any interaction with Rer1p when fused to NubG. This lack of interation is not due to spatial separation between the two proteins as Ost1p-NubI was able to interact with Rer1p-Cub-PLV. This negative control provides additional evidence for the specificity of the interactions observed in the split-ubiquitin system. NubG-Alg5p showed a faint reaction with Rer1p-Cub-PLV, possibly due to a very weak or transient interaction between the two proteins.
This work shows that the transmembrane domain of Mns1p contains an ER localization signal recognized by Rer1p. Furthermore, the split-ubiquitin system demonstrates the interactions of Mns1p and Sec12p with Rer1p in vivo. These data constitute further evidence that Rer1p is at the center of a retrieval system mediated by the transmembrane domain of several ER proteins (Massaad et al., 1999; Sato et al., 1997; Sato et al., 2001; Sato et al., 1996). Prior to this study, the topology of Rer1p was based on hydrophobic sequence analysis (Boehm et al., 1994; Sato et al., 1995). The split-ubiquitin system demonstrates that the C-terminal of Rer1p, where Cub has been linked, is in the cytosol. The split-ubiquitin system is therefore useful for studying the interaction as well as the topology of membrane proteins in their native environment.
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ACKNOWLEDGMENTS |
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