The Sec61 protein is required for protein translocation across the ER membrane in both yeast and mammals and is found in close association with polypeptides during their membrane transit. In Saccharomyces cerevisiae Sec61p is essential for viability and the extent of sequence similarity between the yeast and mammalian proteins (55% sequence identity) suggests that the role of Sec61p in the translocation mechanism is likely to be conserved. In order to further our understanding of the structure and function of Sec61p we have cloned homologues from both Schizosaccharomyces pombe and Yarrowia lipolytica. The S. pombe gene comprises six exons encoding a 479 residue protein which we have immunolocalised to the endoplasmic reticulum. Sequence comparisons reveal that S. pombe Sec61p is 58.6% identical to that of S. cerevisiae. The deduced amino acid sequence of the Y. lipolytica protein shares 68.8% sequence identity with S. cerevisiae Sec61p.
Gene disruption studies have shown that the SEC61 is required for viability in both S. pombe and Y. lipolytica demonstrating that the essential nature of this protein is not unique to S. cerevisiae. Moreover, heterologous complementation studies indicate that the Y. lipolytica SEC61 gene can complement a null mutation in S. cerevisiae. Sequence comparisons between the various eukaryotic Sec61p homologues reveal a number of highly conserved domains, including several transmembrane sequences and the majority of cytosolic loops. These comparisons will provide an important framework for the detailed analysis of interactions between Sec61p and other components of the translocation machinery and between Sec61p and translocating polypeptide chains.
Current evidence suggests that protein translocation across the membrane of the endoplasmic reticulum (ER) occurs via an aqueous channel (Simon and Blobel, 1991; Crowley et al., 1993, 1994). A variety of crosslinking studies indicate that the integral membrane protein, Sec61p, is in close proximity to various polypeptides during their membrane translocation suggesting that Sec61p contributes, at least in part, to the formation of the translocation channel (Sanders et al., 1992; Müsch et al., 1992; Görlich et al., 1992; High et al., 1993; Mothes et al., 1994; Oliver et al., 1995; Hanein et al., 1996). SEC61 was first identified in Saccharomyces cerevisiae via the isolation of mutants defective in the translocation of both secretory and integral membrane proteins into the ER (Deshaies and Schekman, 1987; Stirling et al., 1992). The Sec61 protein (Sec61p) is a 53 kDa integral membrane protein whose transmembrane topology has recently been determined and has been shown to span the bilayer ten times (Stirling et al., 1992; Wilkinson et al., 1996). Interestingly, several transmembrane domains have very limited hydrophobic characteristics consistent with them participating in the formation of the aqueous environment reported for the translocation site. A mammalian homologue of Sec61p, termed Sec61α (Görlich et al., 1992), has been purified as a complex with two other proteins, Sec61β and Sec61γ, and this complex is required for the reconstitution of protein translocation into proteoliposomes (Görlich and Rapoport, 1993). A homologous trimeric ‘Sec61-complex’ has also been identified in yeast comprising Sec61p (Sec61α), Sbh1p (Sec61β), and Sss1p (Sec61γ) (Esnault et al., 1994; Hartmann et al., 1994; Panzner et al., 1995). In S. cerevisiae both Sec61p and Sss1p are encoded by essential genes (Stirling et al., 1992; Esnault et al., 1993). In contrast, SBH1 is non-essential and a null mutant has no detectable defect in protein translocation (Finke et al., 1996), thus the role of the β-subunit of the Sec61-complex remains unclear. More recently a Sec61p homologue, Ssh1p, has been identified in yeast that is 32% identical to Sec61p itself (Hølmstrom et al., 1994). Intriguingly, Ssh1p is found to form a second trimeric complex comprising a further Sec61β homologue, Sbh2p, together with Sss1p (Finke et al., 1996). Neither Ssh1p nor Sbh2p are essential for viability in yeast, and there is currently no clear evidence that this second trimeric complex is involved in translocation.
Sequence comparisons reveal that the essential components of the Sec61-complex are highly conserved. Yeast Sss1p is 44% identical to mammalian Sec61γ and the two proteins have been shown to be functionally interchangeable in yeast (Hartmann et al., 1994). Sec61p is also highly conserved with 55% sequence identity between the yeast and mammalian proteins (Stirling et al., 1992; Görlich et al., 1992). Further homologues have also been identified in the cryptomonad alga Pyrenomonas salina (Muller et al., 1994), and in the ascidian Halocynthia roretzi (Ueki and Satoh, 1994), however, it is unclear whether these homologues are functionally equivalent to yeast Sec61p, or whether they may be functionally redundant as in the case of Ssh1p.
In order to refine our understanding of the structure and function of Sec61p we have sought to identify homologues from two distantly related eukaryotes, namely Schizosaccharomyces pombe and Yarrowia lipolytica. Both of these organisms are amenable to reverse genetic analysis and therefore offer the prospect of examining the role played by any novel Sec61 homologues in vivo. Our studies have led to the identification of a single gene from each organism which we have found to be essential for viability in their respective systems. Furthermore, we have exploited these novel homologues to address the extent of functional conservation amongst Sec61 proteins. Here we demonstrate that the Y. lipolytica SEC61 can functionally complement a sec61 null mutation in S. cerevisiae, whereas the S. pombe homologue cannot. Comparisons between these various Sec61p sequences, coupled with the insights gained from the study of functional equivalence, now provide powerful insights into the structure and function of this essential component of the ER translocase.
MATERIALS AND METHODS
Restriction enzymes, modifying enzymes, ampicillin, and dNTPs were from Boehringer Mannheim. Tunicamycin, and DAPI were from Sigma. Yeast lytic enzyme and [α-32P]dCTP were from ICN. Culture media were obtained from Difco.
Strains and growth conditions
Escherichia coli and yeast strains used are listed in Table 1. E. coli cells were grown in LB at 37°C (1% tryptone, 0.5% yeast extract, 1% NaCl). Where appropriate, ampicillin was used at a final concentration of 100 μg ml−1. S. cerevisiae strains were routinely grown in YPD (2% peptone, 1% yeast extract, 2% glucose) or in YNB (0.675% yeast nitrogen base) with 2% glucose and appropriate amino acid supplements. URA3+ cells were counterselected using 5-fluoroorotic acid (FOA)-containing medium (Sikorski and Boeke, 1991). Solid medium was supplemented with 2% Bacto agar. Diploids were sporulated on 1% KOAc, 0.1% yeast extract, 0.05% glucose plus appropriate amino acid supplements, at 24°C. Tetrad dissection was as described by Sherman and Hicks (1991). Yarrowia lipolytica was grown routinely at 28°C in YPDYl medium (1% yeast extract, 1% bactopeptone, 1% glucose), or in minimal medium (0.17% yeast nitrogen base without amino acids, 1% glucose, 0.1% proline) with 0.01% amino acids. Schizosaccharomyces pombe strains were grown routinely at 30°C in rich YPD medium or in minimal EMM2 media (Gutz et al., 1974; Moreno et al., 1990). Mating and meiotic induction were performed as described by Hagan and Yanagida (1995).
DNA manipulation techniques
Standard techniques have been used according to the method of Sambrook et al. (1989). S. pombe total DNA was prepared essentially as described by Moreno et al. (1990). Y. lipolytica DNA was extracted by the method of Hoffman and Winston (1987). Plasmids were transformed into S. cerevisiae using the lithium acetate method described by Ito et al. (1983). Yarrowia lipolytica transformations were carried out according to the method of Davidow et al. (1985) as modified by Xuan et al. (1990). S. pombe transformations were performed as previously described (Moreno et al., 1990).
Amplification of Schizosaccharomyces pombe SEC61 gene fragment by PCR
A DNA fragment was amplified by PCR from S. pombe genomic DNA using degenerate primers SpA (forward): 5′ AAG GGI TWC GGI CTI GGI KCI GGI ATT 3′; SpB (reverse): 5′ IGT RTA RAA IAR YTT IAT IGG RTA 3′; and SpC (reverse): 5′ YTC IAT CCA IGT YTT ISW RAA 3′, corresponding to three regions conserved between canine and S. cerevisiae Sec61 proteins SpA: KGYGLGSGI; SpB: YPIKLFYT; SpC: FSKTWIE. Amplifications were performed over 40 cycles in a Techne PHC-3 thermal cycler as follows: denaturation, 1 minute, 94°C; annealing, 2 minutes, 50°C; elongation, 2 minutes, 72°C, with a ramp rate of 10°C per minute between annealing and elongation.
Amplification of Yarrowia lipolytica SEC61 gene fragment by PCR
A Yarrowia lipolytica SEC61 gene fragment has been obtained by PCR on Y. lipolytica (W29) genomic DNA: denaturation, 30 seconds, 94°C; hybridisation, 30 seconds, 46°C; elongation, 1 minute, 72°C; 25 cycles (Hybaid, CERA LABO). Primers YlA (forward): 5′ AAG GGT TAC GGC CTG GGT TCT GGT ATC TC 3′; and YlB (reverse): 5′ GTT AGA GGT GTA GAA CAG CTT GAT GGG 3′ used for the amplification were designed according to codon usage of Yarrowia lipolytica and corresponding to two amino acid sequences conserved between canine and Saccharomyces cerevisiae Sec61 proteins (Stirling et al., 1992; Görlich et al., 1992): YlA: KGYGLGSGIS and YlB: PIKLFYTSN.
Y. lipolytica genomic DNA library
A Yarrowia lipolytica LEU2-based genomic library (M. Chasles, P. Fournier and C. Gaillardin, unpublished), was screened with the Yarrowia lipolytica SEC61 gene fragment obtained by PCR after labelling with [α-32P]dCTP (ICN) by random priming (Boehringer Mannheim). Hybridization and washing were carried out essentially as described by Sambrook et al. (1989).
A 3.5 kbp EcoRV fragment of S. pombe genomic DNA containing the full length SEC61 gene was cloned into the SmaI site of pUC118 in both orientations to create pJBS5 and pJBS5.1, respectively. These plasmids and various subcloned derivatives were used to produce single stranded DNA for use as templates in DNA sequencing reactions as described above. A sec61Sp null allele was constructed by first introducing an NdeI restriction site at the 3′ end of the SEC61Sp coding sequence by site directed mutagenesis essentially as described by Kunkel et al. (1987). Single stranded template DNA from pJBS5.1 was mutated using oligonucleotide SDM1: 5′ GTTACTCC-CATATGGCAATAA 3′ to create pJBS5.2. This engineered site was then used in conjunction with the naturally occurring NdeI restriction site close to the 5′ end of the coding region to remove the bulk of the SEC61 coding sequence. A 1.8 kbp HindIII fragment from plasmid pIH5 (supplied by Iain Hagan) containing the S. pombe URA4 gene (Grimm et al., 1988) was ligated into NdeI-digested pJBS5.2 (after both fragments had been filled-in using the Klenow fragment of DNA polymerase I plus dNTPs) to create pJBKO in which URA4 replaces 95% of the translated coding region of the SEC61 gene. The resultant gene disruption cassette was then excised from pJBK0 as an EcoRI/SalI fragment then used to transform S. pombe to uracil prototrophy.
A plasmid expressing an epitope tagged version of S. pombe Sec61p was constructed as follows. Site directed mutagenesis was first used to introduce an NdeI restriction site into the genomic copy of S. pombe SEC61 in pJBS5.1 using the oligonucleotide SDM2 (5′-TTAC-CCGTTACTCCCCATATGCAATAAAATGCC 3′) to create pJBT1.
This mutation placed the CAT of the NdeI recognition site in register with the SEC61 reading frame and resulted in the change of a methionine to a histidine residue situated three amino acids from the C terminus. A SalI/SacI fragment from pJBT1 corresponding to the mutated SEC61 gene was then subcloned into pSP1 (Cottarel et al., 1993) to create pJBT2. A 113bp NdeI fragment encoding two tandem c-myc 9E10 epitopes (Evan et al., 1985; Craven et al., 1996) was then ligated into pJBT2 which had been linearised by partial digestion with NdeI. Clone pJBT3 was shown by DNA sequencing to contain a single inframe insertion of the epitope encoding fragment in the NdeI site at the 3′ end of SEC61.
Plasmid pYL1078 was isolated from a plasmid library of Y. lipolytica genomic DNA and was found to contain a 5.9 kbp insert including the complete SEC61Yl gene. A gene disruption cassette was then constructed by cloning a 1.7 kbp BglII/BamHI fragment from pINA490 containing the Y. lipolytica URA3 gene into the unique BamHI site in pYL1078 to create pYL1175. A 5 kbp ClaI fragment from pYL1175 containing the sec61Yl::URA3 cassette (plus 350 bp of vector sequence) was then used to transform Y. lipolytica to uracil prototrophy.
Expression of Sec61Yl in Saccharomyces cerevisiae
First an expression vector with a suitable marker was created by deleting the URA3 gene (as a BglII fragment) from pFL61 (Minet et al., 1992) and replacing this with the S. cerevisiae TRP1 gene from YRp7 (Struhl et al., 1979) as a BglII/EcoRI fragment inserted between BamHI and EcoRI to produce pYL1043. The Y. lipolytica SEC61 coding sequence was then amplified by PCR with a NotI site on both sides and cloned into pYL1043 restricted with NotI to produce pYL1044 (the SEC61 coding region was checked by DNA sequencing). This plasmid has the SEC61Yl coding sequence cloned under the regulation of the S. cerevisiae PGK promoter and terminator elements. A HindIII/XbaI fragment from pYL1044 (PGK-SEC61Yl) was cloned into the centromeric plasmid pRS315 (Sikorski and Hieter, 1989) to produce pYL1174.
Cloning the S. pombe SEC61 cDNA into an S. cerevisiae expression vector
A full length cDNA clone (pJBC1) was isolated from the Norbury cDNA library. The insert in pJBC1 was cloned as an SphI (end repaired with Klenow fragment)/SalI fragment into the XhoI (end repaired with Klenow fragment and dNTPs)/SalI sites of the yeast expression vector pKL3. The resultant construct, pJBT6, places the SEC61SpcDNA under the control of the ADH promoter/terminator in a CEN4, ARS1, TRP1 vector.
Yarrowia lipolytica SEC61 gene was subcloned in pBluescript SK+ and sequenced by primer walking on a fluorescent DNA sequencer (ABI Biosystems-Perkin Elmer) according to the supplier. Sequence data were analysed with the Sequence Assembly Program XBAP (Dear and Staden, 1991, 1992). The S. pombe SEC61 gene was sequenced by primer walking using single stranded DNA template according to the methods described by Stirling et al. (1992). Sequence assembly, manipulation and multiple sequence comparisons were performed using the UWGCG package (Devereux, 1994).
Whole cell extracts and western blotting
Indirect immunofluorescence in S. pombe
This was performed essentially as described by Hagan and Hyams (1988).
Identification of a SEC61 homologue in Schizosaccharomyces pombe
Consensus protein sequences for the design of degenerate PCR primers were derived from a multiple alignment of S. cerevisiae Sec61p, mammalian Sec61α, plus SecY from the archaebacteria M. vanneilii (Auer et al., 1991) and H. marismortui (Arndt, 1992) (see Materials and Methods). PCR reactions using these primers generated a product of the expected size (627 bp) when using S. cerevisiae genomic DNA as template, but led to a single slightly larger product from S. pombe. Nested PCR using a reverse primer internal to the product (see Materials and Methods) and the original forward primer yielded products of the expected size when first round PCR products from both yeast species were used as the template, suggesting that the first round PCR products were indeed authentic (data not shown). The nucleotide sequence of the large PCR product from S. pombe demonstrated a close sequence similarity to the SEC61 gene from S. cerevisiae. That the amplified sequence represented a single genetic locus in S. pombe was demonstrated by Southern blotting of digested genomic DNA using radiolabelled PCR product as the probe (data not shown). The same probe was then used to identify full length S. pombe SEC61 clones from both genomic and cDNA libraries. The probe was found to hybridise to the genomic cosmid clone (21D6c) whose insert has been mapped near to the mei3 marker on chromosome II (Maier et al., 1992). Southern blotting analysis was used to compare the restriction maps of the SEC61 locus in genomic DNA versus cosmid 21D6c, and to identify fragments to be subcloned into pUC118 for the purpose of sequence determination. Multiple rounds of sequencing using sequential primers allowed the complete double strand sequence of a 2,366 base pair portion of S. pombe genomic DNA to be determined (data not shown). Analysis of the coding potential of this sequence suggested that the S. pombe SEC61 gene might contain several introns. Therefore, in order to confirm these predictions the sequence of a full length cDNA (pJBC1) was obtained. This sequence contained a single long open reading frame that would encode a 479 residue polypeptide with a predicted molecular mass of 53.3 kDa (Fig. 1A). The deduced amino acid sequence of this protein shares 58.6% sequence identity with Sec61p from S. cerevisiae (with a total of 75% sequence similarity when conservative substitutions are considered (see Fig. 2). Henceforth we refer to this gene as SEC61Sp.
Comparison of the genomic and cDNA sequences confirmed the presence of five introns clustered towards the 5′ end of the gene. These introns vary in length from 42-177 bp with the shortest exon being 33 bp in length. The complete genomic sequence of S. pombe SEC61, including full details on the intron/exon boundaries, has been submitted to EMBL (accession number Y11375).
Cloning of SEC61 from Y. lipolytica
Using a similar approach we have obtained a PCR product from Y. lipolytica genomic DNA which was then used as a probe to identify candidate clones from a Y. lipolytica (W29) genomic DNA library. Restriction analysis of one such clone, pYL1078, revealed an insert of approximately 5.9 kbp which was then subcloned into pBluescript SK+ for DNA sequence determination (see Materials and Methods). The complete double stranded sequence of a 2,185 bp region was obtained and found to contain a 471 codon open reading frame which would encode a polypeptide with a predicted molecular mass of 51.7 kDa (Fig. 1B). This deduced protein sequence is strikingly similar to that of S. cerevisiae Sec61p with 68.8% sequence identity (84% sequence similarity overall when conservative substitutions are included; Fig. 2). We refer to this gene as SEC61Yl.
The S. pombe SEC61Sp gene is essential for viability
As a first step in the functional analysis of Sec61p in S. pombe we have created a null mutant by targeted gene disruption as follows. The full length genomic copy of S. pombe SEC61 was first subcloned as a 3.5 kb EcoRV fragment in pJBS5.1, then used to construct a sec61 null allele in which 95% of the SEC61 coding sequence has been replaced with the S. pombe URA4 gene to create a sec61::URA4 null allele (Fig. 3). The chimeric EcoRV fragment (containing the sec61::URA4 allele) was used to transform a diploid S. pombe strain IH310 to uracil prototrophy. Genomic DNA from several transformants was isolated and subjected to Southern blot analysis to assess the location of integration of the sec61::URA4 allele (Fig. 3B). In three out of twenty Ura+ transformants analysed the sec61::URA4 fragment was found to have integrated at the SEC61 locus to create authentic heterozygous diploids (SEC61/sec61::URA4). Sporulation and tetrad dissection of one such diploid (JBKO15) resulted in no more than two viable spores from any tetrad with no viable Ura+ progeny. In contrast, when strain JBKO15 was transformed with a plasmid containing a functional genomic copy of S. pombe SEC61 (pJBT3, Leu+), 5 out of 29 tetrads contained 3 viable spores, and one contained 4 viable spores. In this case viable Ura+ spores were recovered (indicating a disrupted sec61::URA4 genotype) but these were always Leu+ indicating the presence of the plasmid pJBT3. After prolonged propagation of these Ura+/Leu+ (sec61::URA4 plus pJBT3) cells in rich medium all viable clones were shown to retain pJBT3. This contrasts with the high degree of instability observed for pJBT3 when Ura−/Leu+ spores (i.e. SEC61 plus pJBT3) were grown under non-selective conditions, where 27% of colony forming units were plasmid free after a single subculture in rich medium. Taken together these data indicate that SEC61 is essential for vegetative growth in S. pombe and that pJBT3 encoding an epitope tagged version of Sec61p (see Materials and Methods) can functionally complement the sec61::URA4 null mutation.
SEC61Yl is essential for viability in Yarrowia lipolytica
A 1.7 kb BglII-BamHI fragment containing the Yarrowia lipolytica URA3 gene was isolated from plasmid pINA490 and inserted into the BamHI site within the coding sequence of Y. lipolytica SEC61 to create a disrupted allele (Fig. 4). A ClaI fragment containing this disruption cassette was used to transform a Yarrowia lipolytica haploid strain (INAG1236463) carrying a replicative plasmid with the wild-type copy of SEC61 gene. Ura+ transformants were selected and screened for their inability to grow on 5-FOA medium to counterselect integration in the replicative plasmid. Then candidates were checked by PCR and Southern blotting to confirm the site of integration of the disrupted sec61::URA3 allele (data not shown). The disrupted strain, YLKO1 was tested for the segregation of the replicative plasmid carrying the wild-type copy of Yarrowia lipolytica SEC61 gene after 25 generations in YPD medium. No viable plasmid free clones were obtained from which we conclude that SEC61 is essential for viability in this organism.
Sec61Yl can complement a sec61 null mutation in S. cerevisiae
In order to examine the degree of functional conservation amongst various Sec61p homologues we have sought to express the S. pombe and Y. lipolytica proteins in Saccha-
The SEC61Yl coding sequence was first placed under the control of the yeast PGK promoter/terminator in plasmid pYL1044 (2 μm, TRP1). This plasmid was then tested for its ability to complement a sec61 null mutation in strain BWY47. This strain carries a null allele of sec61 (sec61::HIS3) which can be complemented by a conditional expression allele of SEC61 on plasmid pBW62 (CEN, URA3, GAL-SEC61;Wilkinson et al., 1996). BWY47 (+pBW62) is viable on galactose medium but cannot form colonies on glucose medium due to the transcriptional repression of the GAL promoter (see Fig. 5A). When pYL1044 was transformed into BWY47 (+pBW62) the resultant strain is capable of growth on glucose indicating that the Sec61Yl protein expressed from pYL1044 can function in S. cerevisiae (Fig. 5A). In order to eliminate the possibility that low level expression from pBW62 might contribute to viability on glucose the doubly transformed strain was passaged on 5-FOA medium to select for loss of pBW62. Colonies resistant to 5-FOA were obtained when BWY47 (+pBW62) carried pYL1044 (2 mM, TRP1, PGK-SEC61Yl) or pBW11 (LEU2,CEN, SEC61) (Wilkinson et al., 1996), but not when transformed with a vector only control (data not shown).
In order to test whether S. pombe Sec61p could also function in S. cerevisiae we first transformed BWY47 (+pBW62) with the full length genomic copy of SEC61Sp in either a single copy (pJBS6; LEU2, CEN, SEC61Sp) or multicopy (pJBS7; LEU2,2 μ?, SEC61Sp) plasmid. Suitably transformed cells remained sensitive to glucose indicating that the genomic copy of S. pombe could not rescue the lethal phenotype associated with depletion of Sec61p (data not shown). Since the failure to complement might be related to poor expression from the S. pombe gene (e.g. due to poor transcriptional initiation or incorrect intron processing in the heterologous host) we therefore cloned the S. pombe cDNA into a yeast expression vector. Plasmid pJBT6 contains the full length SEC61Sp cDNA under the control the S. cerevisiae ADH promoter/terminator sequences (see Materials and Methods), but as with the genomic clone, this plasmid was unable to complement the S. cerevisiae sec61 null mutation (Fig. 5A).
The data described above clearly demonstrate that Sec61Ylrelates with a slight accumulation of translocation precursors in BWY47 carrying pYL1044 (Fig. 5C).
Immunodetection of S. pombe Sec61p
To visualise the S. pombe Sec61 protein an epitope tag was appended to the carboxy terminus by gene fusion (see Materials and Methods). The tagged protein was shown to be functional by its ability to complement the sec61::URA4 disruption described above. Whole cell extracts were prepared from S. pombe strains carrying these constructs, for analysis by western blotting. It has previously been reported that S. cerevisiae Sec61p aggregates on incubation at 100°C (Stirling et al., 1992; and unpublished data). Accordingly, S. pombe whole cell extracts were prepared by mechanical disruption in Laemmli sample buffer (Laemmli, 1970) and then incubated at either 0, 50 or 100°C prior to SDS-PAGE analysis. Western blotting analysis using 9E10 anti-cMyc monoclonal antibodies identified a single polypeptide band in cells carrying plasmid protein can complement a sec61 null mutation in S. cerevisiae, but the following observations indicate that the extent of functional equivalence is not complete. Firstly, BWY47 + pYL1044 has a slightly increased doubling time when compared to wild type (Fig. 5B). This reduction in growth rate might be due to a detrimental effect of the overexpression of Sec61Yl protein from the PGK expression vector but this seems unlikely given that pYL1044 has no impact on growth rate in a wild-type yeast strain (data not shown). The reduction in growth rate also cor-pJBT3 encoding the c-Myctagged copy of SEC61 (Fig. 6A). The immunoreactive band was detectable in extracts prepared at either 0 or 50°C (Fig. 6A, lanes 2 and 3), but was not in extract prepared at 100°C (Fig. 6A, lane 4). This observation would be consistent with aggregation of the Sec61 protein at high temperature. Whilst the deduced primary sequence of the c-Myc tagged Sec61 protein predicts a molecular mass of 53.3 kDa, its gel mobility indicates a relative molecular mass of 40 kDa.
Localisation of Sec61p in S. pombe
The epitope tagged Sec61p construct described above was used in the localisation of Sec61 protein to structures resembling the ER. Indirect immunofluorescence microscopy using the 9E10 monoclonal antibodies reveal strong perinuclear staining with strands radiating from the central ring structure towards the poles of the cell. There was often a distinct increase in the density of diffuse staining towards the poles in non-dividing cells (see Fig. 7A and C). This specific staining pattern was overlaid with bright punctate staining which was not specific to the tagged Sec61p since this pattern was also seen in cells expressing only untagged Sec61Sp (Fig. 7B). The level of staining varies from one cell to another which we interpret as a likely consequence of variations in plasmid copy number from cell to cell. Despite differences in the intensity of staining between cells, the qualitative pattern remains similar suggesting that overexpression of Sec61p does not contribute to gross mislocalisation in this system. The immunofluorescence staining pattern seen for Sec61p is consistent with that seen for the S. pombe BiP homologue which has been localised by immunofluorescence microscopy of a Myc-tagged protein (Pidoux and Armstrong, 1992) and subsequently by using anti-bodies raised to the BiP protein itself (Pidoux and Armstrong, 1993). In both cases the antigen localised to a perinuclear ring, cytosolic strands and a peripheral reticular network. This pattern is characteristic of the endoplasmic reticulum in mammalian cells and is also very similar to that seen in S. cerevisiae (Deshaies and Schekman, 1990; Feldheim et al., 1992).
In this report we have described the cloning and characterisation of SEC61 homologues from two distantly related species, Schizosaccharomyces pombe and Yarrowia lipolytica. The S. pombe gene, SEC61Sp, comprises 6 exons encoding a 479 amino acid residue polypeptide that is 58.6% identical to S. cerevisiae Sec61p and 57% identical to mammalian Sec61α. The Y. lipolytica gene, SEC61Yl, contains a single exonencoding a 471 residue polypeptide which is 68.8% identical to S. cerevisiae Sec61p. The Y. lipolytica protein also shares 63.8% identity with the S. pombe homologue and 60% identity with mammalian Sec61α. The multiple sequence comparison shown in Fig. 2 illustrates the degree of sequence conservation amongst known eukaryotic Sec61 proteins. The inclusion of these two novel sequences refines the comparison substantially with only 151 residues being absolutely conserved in all Sec61 proteins listed, of which only 76 residues are also conserved in the functionally redundant Ssh1p. Clearly, Ssh1p remains by far the most divergent Sec61p homologue (33% sequence identity between the two S. cerevisiae proteins) but the significance of this comparison is uncertain given that any role for Ssh1p in protein translocation in S. cerevisiae remains to be established (see below).
The S. cerevisiae Sec61 protein has been shown to span the ER membrane 10 times (Wilkinson et al., 1996). The hydropathy profiles of the S. pombe and Y. lipolytica proteins are remarkably similar to that predicted for S. cerevisiae Sec61p, from which it would appear likely that they would adopt a very similar transmembrane topology (Fig. 8). One interesting point raised by these comparisons is the conservation of a hydrophobic sequence between residues 175-200 located between transmembrane domains 4 and 5. The extent of conservation within this short domain suggests that it may be crucial to Sec61p function, however, it is unclear whether or not this domain actually spans the bilayer. Data from Wilkinson et al. (1996) indicates the presence of only two transmembrane domains between residues 175 and 276 in S. cerevisiae Sec61p, but the identification of the short hydrophobic segment between residues 213 and 224 as ‘transmembrane domain 5’ may be incorrect. It seems likely that one of the two hydrophobic domains between residues 175 and 225 actually spans the bilayer whilst the other may either be embedded in the membrane, or may be involved in hydrophobic interactions with another protein. This latter role might be expected to require the degree of sequence conservation observed between residues 175 and 200.
A second interesting feature of the multiple comparison is the high degree of sequence conservation in transmembrane domain 2 (TM2). This domain is of particular interest since it contains several relatively hydrophilic residues and has been shown to require an interaction with TM3 for its stable integration into the bilayer. These features have led to the prediction that TM2 might contribute to the creation of a hydrophilic surface lining the interior of the translocation channel (Wilkinson et al., 1996). The conservation of several hydrophilic residues within TM2 would lend support to this hypothesis. Intriguingly, there is no obvious conservation of complementary charged residues in TM3 as might be expected if salt bridges between the two domains were involved in the observed interaction between TM2 and TM3. This is somewhat unexpected and may indicate that TM3 does not interact directly with TM2, but that it assists in recruiting some trans-acting factor which then stabilises TM2 in the bilayer. This proposal will require further study. The significance of sequence conservation within TM2 is further highlighted by the fact that this is the most conserved transmembrane domain between Sec61p and bacterial SecY suggesting that it plays a crucial role in the structure and function of the Sec61/SecY family of translocases (Stirling et al., 1992; Görlich et al., 1992; Stirling, 1993).
Other notable conserved regions amongst Sec61p homologues include several domains oriented towards the cytosolic surface of the ER membrane, including the loops located between TM4-TM5 (although see above), TM6-TM7, and TM8-TM9. These conserved domains may be involved in specific interactions with components of cytosolic protein translation/targeting machinery, or with cytosolic domains of other ER membrane proteins involved in targeting/translocation. Indeed, the conservation within loops TM6-7 and TM8-9 would be consistent with the recent demonstration that this region of Sec61p is involved in the stable association of Sss1p (Wilkinson et al., 1997).
The extent of sequence similarity observed between the various Sec61p homologues closely mirrors the phylogenetic distances between the species. Accordingly, Sec61p from Y. lipolytica is most similar to that of S. cerevisiae, whilst that of S. pombe is only slightly closer to S. cerevisiae than to mammals. The Y. lipolytica Sec61 protein is 69% identical (and 84% similar) to the S. cerevisiae sequence and can functionally complement the lethal phenotype associated with a null mutation in S. cerevisiae. This represents the first demonstration of functional conservation in this core component of the ER translocation machinery. Yet despite the very high degree of sequence similarity the two proteins are clearly not equivalent since S. cerevisiae cells expressing Sec61Yl protein have a retarded growth rate and accumulate a proportion of preproCPY in an untranslocated form. This partial complementation may reflect the very complex interactions required between Sec61p and the other factors involved in the translocation process. Indeed, it may be that the differences between the S. cerevisiae and Y. lipolytica sequences may prove to be as informative as their similarities. Moreover, intra- and/or extragenic suppressors of the partial complementation phenotype may identify critical domains involved in the quaternary structure and function of this essential protein. The failure of the S. pombe SEC61 gene to complement in S. cerevisiae would seem likely to be related to the gene’s complex intron structure. However, even the cDNA failed to complement when cloned into a suitable expression vector under the control of the ADH promoter. Clearly, such negative data must be interpreted with caution but, given the partial complementation by the more closely related Sec61pYl, it would appear probable that the S. pombe protein has diverged to such an extent that it can no longer interact productively with one or more components of the S. cerevisiae translocation machinery. This present study does not directly address the role of Sec61p in protein translocation in S. pombe, but the degree of sequence similarity with other Sec61p homologues, coupled with the essential nature of the SEC61Sp gene, and the ER localisation of the Sec61pSp, make it seem highly probable that this protein will prove to be involved in translocation across the ER membrane in this organism.
Studies in S. cerevisiae have shown SEC61 to be an essential gene whose product is involved in both co- and trans-postlational translocation of secretory and membrane protein pre-cursors (Deshaies and Schekman, 1987; Stirling et al., 1992; Panzner et al., 1995; Ng et al., 1996). In contrast SSH1 is functionally redundant but the fact that it can be purified as a component of a trimeric complex (containing Sss1p and Sbh2p) is highly suggestive of a role as a protein translocase. Moreover, the observation that this Ssh1 complex is found associated with ribosomes but not the Sec62/63 complex has led to the suggestion that any role for Ssh1p in translocation might be restricted to the co-translational mechanism (Finke et al., 1996). Should this indeed prove to be the case then it raises intriguing questions as to the essential nature of Sec61p. Perhaps significantly yeast Sec61p has been shown to be involved in post-translational translocation as a component of a heptameric complex comprising Sec62p and Sec63p. Interestingly, yeast Sec62p and Sec63p are both encoded by essential genes raising the possibility that the essential function of Sec61p might be related to its role in post-translational translocation. In this study we have identified a single Sec61 homologue in two distantly related yeast species. In both cases these homologues are more closely related to Sec61p than to Ssh1p but our analysis does not preclude the existence of Ssh1-like proteins in these species since our PCR-based strategy would have been biased against the identification of such genes. The finding that sec61 null alleles are lethal in both S. pombe and Y. lipolytica is significant since it demonstrates that the essential function of Sec61p is not restricted to S. cerevisiae. Moreover, the finding that SEC61Yl can complement a null mutation in S. cerevisiae confirms that the essential function is itself conserved. Characterising the role, if any, of Ssh1p in protein translocation in S. cerevisiae, plus the identi-fication of similar redundant complexes in other systems, will now be essential for us to finally disentangle the complex puzzle surrounding the relative importance of the co- and post-translational translocation mechanisms in vivo.
Thanks are due to Iain Hagan for his advice on S. pombe genetics and for providing strains, reagents, and access to fluorescence imaging equipment. Also to Chris Norbury for providing the S. pombe cDNA library, to Elmar Maier for S. pombe genomic cosmid filters and clones, and to Greg Steele for providing 9E10 antibody and Martin Watson for anti-CPY antibodies. J.B. was supported by a Glaxo Studentship. This work was further supported by the European Union DGXII. C.J.S. is a Lister Institute Jenner Research Fellow.