1 Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finland
2 Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland
* Author for correspondence (e-mail: marja.makarow{at}helsinki.fi)
Accepted 5 September 2003
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Summary |
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Key words: Yeast, ER, Golgi, Glycosyltransferases, Activity, Recycling
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Introduction |
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The protein-bound O-glycans of S. cerevisiae consist of linear arrays of up to five mannose residues. Their assembly is initiated in the ER by protein mannosyltransferases Pmt1p and Pmt2p on serine and threonine residues of newly translocated proteins (Strahl-Bolsinger et al., 1993; Lussier et al., 1995a). Then, an
1,2-linked residue is added, and to it another one by
1,2-mannosyltransferase Mnt1p in the Golgi (Haselbeck and Tanner, 1993
; Häusler et al., 1992
; Lussier et al., 1995b). Finally, two successive
1,3-linked mannose residues are added by Mnn1p, the same transferase that terminates the branches of N-glycans.
The Golgi glycosyltransferases are type 2 transmembrane proteins with a short cytosolic N-terminal fragment. They have been thought to be resident proteins of Golgi subcompartments. Och1p and the M-Pol I and M-Pol II complexes are believed to reside in the cis-Golgi (Jungmann and Munro, 1998; Jungmann et al., 1999
), and Mnn1p in the medial/trans-Golgi (Lussier et al., 1995b; Harris and Waters, 1996
). Thus, decorations by these enzymes have been generally taken as an indication of arrival of the substrate protein in the Golgi. As resident ER proteins lack Golgi-specific glycan decorations, newly synthesized transferases en route to the Golgi have been thought not to function in the ER.
However, recently it was shown that components of M-Pol I and M-Pol II recycle back and forth between the Golgi and the ER. The complexes were found to be incorporated into COPII and COPI vesicles in vitro (Todorow et al., 2000). Exit of membrane-bound and soluble proteins from the ER occurs in vesicles whose cytosolic face is covered with the COPII coat consisting of four structural proteins, whereas Golgi-derived vesicles recycling proteins back to the ER are covered with the COPI coat assembled from an unrelated set of seven proteins (Barlowe, 1998
). We show here that Golgi glycosyltransferases were able to extend N- and O-glycans on newly synthesized exocytic proteins, which were blocked in the ER in COPII mutants. Relocation of Och1p from the Golgi to the ER in COPII-defective mutants was demonstrated by indirect immunofluorescence. Our data suggest that O-glycosylating enzymes travel back and forth between the Golgi and the ER, and that both they, and Och1p, do function in the ER when allowed to accumulate there together with substrate proteins.
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Materials and Methods |
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Other methods
Metabolic labelling with [35S]methionine/cysteine (1000 Ci/mmol) and 2-[3H]mannose (11.5 Ci/mmol; Amersham International, Buckinghamshire, UK), as well as immunoprecipitation with antisera against Hsp150 (1:400, 2 hours), ß-lactamase (1:100, 2 hours), CPY (1:100, 2 hours), 1,6-mannose residues (1:100, overnight), and monoclonal antibody against pentahistidine (Qiagen, USA; 1:100, overnight), as well as SDS-PAGE in 8% gels were like described before (Paunola et al., 1998
). Quantitation of the radioactive signals was performed using Tina 2.0 software. Lectin precipitation was performed with 0.5% Concanavalin A-Sepharose (Amersham Pharmacia Biotech AB, Uppsala, Sweden) in 20 mM Tris-HCl, pH 7.4, containing 0.2 M NaCl and 2% Triton X-100, overnight at 4°C. The lectin beads were released by boiling for 3 minutes in 1% SDS. Indirect immunofluorescent staining was according to Suntio et al. (Suntio et al., 1999
), with a 1:100 dilution of antiserum against the HA epitope (Santa Cruz, USA), or of monoclonal antibody against opsin (Adamus et al., 1991
). In double staining anti-mouse-Alexa488 and anti-rabbit-Alexa568 (Molecular Probes, USA) were used as secondary antibodies. DAPI, CHX and NaN3 (Sigma, USA) were used in final concentrations of 2 µg/ml, 100 µg/ml and 10 mM, respectively.
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Results |
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To rule out the possibility that the 1,6-mannose decoration of pro-CPY we observed in the ER-blocked molecules could be due to other enzymes than Och1p, we repeated the above experiment using a wild-type strain from which the OCH1 gene had been deleted. Mature CPY was immunoprecipitated with CPY antiserum, but none of it was reimmunoprecipitated with the
1,6-mannose-antiserum (Fig. 1C). The parental strain H245 gave similar results as shown in lanes A2 and B2 for the sec13-1 mutant at permissive temperature 24°C (not shown). We conclude that recycling Och1p molecules were under restrictive conditions in the sec13-1 and sec23-1 mutants trapped in the ER, where they decorated newly synthesized pro-CPY molecules with
1,6-mannose.
To study whether newly synthesized Och1p molecules, which had not yet left the ER when the COPII pathway was blocked, had a role in 1,6-mannose decoration of pro-CPY, the above experiment was performed in a sec18-1 mutant. In these cells at 37°C pro-CPY, together with newly synthesized Och1p, was allowed to leave the ER lumen but was blocked in transport vesicles which could not fuse with the Golgi due to non-functional N-ethyl-maleimide-sensitive factor NSF (Sec18p). At the same time, recycling Golgi transferases were blocked in a different compartment, namely Golgi-derived vesicles. Golgi-to-ER transport was inhibited, because NSF is required for all vesicle fusion events of the secretory pathway. Under these conditions, only 4% of pro-CPY was recognized by the
1,6-mannose antiserum (Fig. 1B, lane 3), as compared to the amount immunoprecipitated with CPY antiserum (Fig. 1A, lane 3). Thus, apparently mostly recycling Och1p molecules, rather than their de novo synthesized counterparts, were responsible for
1,6-mannose decoration of pro-CPY in the ER.
Other glycoproteins acquiring 1,6-mannose residues in the ER
Next, we searched for 1,6-mannose decoration in the ER of other glycoproteins. Sec13-1, sec23-1 and
och1 strains were 35S-labeled after 15 minutes preincubation at 37°C to impose the ER exit block in the COPII mutants. The cells were lyzed and subjected to concanavalin A precipitation, to collect the newly synthesized N-glycosylated proteins. One half of the samples were subjected directly to SDS-PAGE analysis (Fig. 2, uneven lanes). The other half was released from the concanavalin A-Sepharose beads and immunoprecipitated with
1,6-mannose antiserum (even lanes). In the COPII mutants, a subset of similar bands as detected by concanavalin A precipitation alone, was recognized by the
1,6-mannose antiserum, suggesting that they were decorated by Golgi-specific Och1p, though arrested in the ER. In the case of the
och1 deletion strain the quantity of the proteins precipitated by the lectin (lane 5) was much less than in the COPII mutants and migrated in the gel differently, apparently due to lack of extension of primary N-glycans. This set of proteins appeared not to be recognized by
1,6-mannose antiserum (lane 6).
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Extension of O-glycans in the ER
Next we studied whether Golgi-specific O-glycan extension could take place in the ER. To this end we used a fusion protein consisting of a 321 amino acid long N-terminal fragment of the secretory yeast glycoprotein Hsp150 (Hsp150) plus the mature portion of E. coli ß-lactamase (Hsp150
-ß-lactamase). The Hsp150
fragment consists of a signal peptide, subunit I (54 a.a.) and 11 repeats of a 19 amino acid peptide (Simonen et al., 1994
). The signal peptide is lost upon ER translocation, and subunit I cleaved in the late Golgi at a Kex2p recognition site (Simonen et al., 1994
). Twenty percent of the amino acids of the Hsp150
portion are serines and threonines, most of which normally are O-glycosylated, whereas the ß-lactamase portion has no, or very few O-glycans (Jämsä et al., 1995
; Suntio et al., 1999
; Holkeri et al., 1996
). The entire fusion protein lacks N-glycosylation sites (Russo et al., 1992
). The O-glycans of mature authentic Hsp150, secreted to the medium from normal cells, are di-, tri-, tetra- and pentamannosides, occurring in the ratio of 4:1:1:1 (Jämsä et al., 1995
). As the Hsp150
fragment carries most of the glycans of Hsp150, mature Hsp150
-ß-lactamase probably has a similar set of O-glycans as mature Hsp150.
We pulse-labelled the sec13-1 mutant for 5 minutes after 15 minutes preincubation at 37°C to block Hsp150-ß-lactamase in the ER. We confirmed recently biochemically and morphologically that Hsp150
-ß-lactamase does not leave the ER at 37°C in the sec13-1 mutant (Fatal et al., 2002
). Immunoprecipitation with ß-lactamase antiserum revealed in the lysate the cytoplasmic form of 66 kDa plus the primary O-glycosylated ER form of 110 kDa (Fig. 3, lane 1) (Paunola et al., 1998
; Fatal et al., 2002
). No protein was found in the medium (lane 5). The Hsp150 signal peptide confers slow post-translational translocation, and this is why some cytosolic form can be detected after a 5 minutes pulse (Paunola et al., 1998
). With increasing chase time, the cytosolic form disappeared with concomitant increase of the glycosylated form, the migration of which became slower. After a chase of 60 minutes all of the cell-associated form migrated like the mature 145 kDa protein (lane 4). A small amount of Hsp150
-ß-lactamase appeared in the medium (lanes 7 and 8), serving as a marker for the fully glycosylated protein. Next we repeated the above experiment by labelling with [3H]mannose instead of [35S]methionine/cysteine. After a 5 minutes pulse, no signal was detected from the immunoprecipitated cell lysate (lane 9). After chase for 10-60 minutes, protein variants could be detected, which comigrated with the respective 35S-labeled forms, and were more and more 3H-labeled (lanes 10-12). As expected, the untranslocated form of 66 kDa could not be labelled with [3H]mannose. In the sec18-1 mutant Hsp150
-ß-lactamase reached only a size of 120 kDa after an hour of chase (lanes 13-16), indicating that it had incomplete O-glycans. This must have been due to failure of the transferase-carrying Golgi-derived vesicles to fuse with the ER, while Hsp150
-ß-lactamase accumulated in ER-derived vesicles. Thus, the reporter protein indeed acquired more mannose residues upon prolonged residence in the ER when exit was blocked in the sec13-1 mutant. Since the ER form of Hsp150
-ß-lactamase contains subunit I, but the secreted 145 kDa form does not, we suggest that the O-glycans of the 145 kDa form of Hsp150
-ß-lactamase, retained in the ER in the sec13-1 mutant, had been extended up to tri-mannosides, but not further.
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O-glycosylation of HDEL-tagged Hsp150-ß-lactamase
If mannosyltransferases of the Golgi can extend O-glycans in the ER, once they recycle from the Golgi to the ER and remain there together with the substrate protein, then conversely, they should perform the same extensions in the Golgi, if the substrate protein recycles between the ER and the Golgi. To study this, we expressed an HDEL-tagged Hsp150-ß-lactamase variant in normal cells. C-terminally HDEL-tagged proteins exit the ER, but are recognized by the Erd2p receptor, which returns them to the ER (Semenza et al., 1990
). When Hsp150
-ß-lactamase-HDEL was 35S-labeled in normal cells, immunoprecipitation showed that it remained cell-associated (Fig. 4, lane 2), and none could be immunoprecipitated from the medium (lane 1). Most of the cell-associated protein (lane 2) comigrated with Hsp150
-ß-lactamase lacking HDEL, which was mostly secreted to the medium in the same strain (lanes 3 and 4). A small amount of cell-associated Hsp150
-ß-lactamase-HDEL (lane 2) co-migrated with the same variant trapped in the pre-Golgi compartment in the sec18-1 mutant (110 kDa, lane 6). As Hsp150
-ß-lactamase-HDEL probably does not reach the latest Golgi subcompartment where Kex2 protease is located, it was likely to contain subunit I, which adds more than 10 kDa to the molecular mass (Suntio et al., 1999
). Thus, when Hsp150
-ß-lactamase-HDEL was allowed to encounter Golgi transferases multiple times by recycling between the ER and the Golgi, its O-glycans were matured, but apparently not to full length.
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Effect of absence of COPI traffic on Golgi-specific N-glycan extension in the ER
To confirm that recycling rather than de novo synthesized Och1p was responsible for decoration of N-glycans in the ER, we used as a reporter the soluble cell wall protein Scw4p, which is N-glycosylated at a single potential site, and runs in SDS-PAGE like a 66 kDa protein (Cappellaro et al., 1998). A SCW4 variant encoding a C-terminally histidine-tagged version of the protein was integrated into the genome of control cells and sec mutants. The control cells were preincubated for 15 minutes at 37°C and 35S-labeled for 5 minutes, and a parallel sample was thereafter chased for 30 minutes. Immunoprecipitation with antibody against pentahistidine revealed a protein migrating at 59 kDa (Fig. 5, lane 1). During chase it was converted to a 66 kDa form (lane 2), apparently the mature form arisen by glycan extension during transport to the cell wall. In the presence of tunicamycin (TM), which inhibits N- but not O-glycosylation, the increase in apparent molecular weight of Scw4p after chase was small (lane 4), probably resulting from extension of some O-glycans during secretion. The above experiment was repeated in a sec23-1 mutant to block Scw4p in the ER. During the chase the apparent molecular weight increased from 59 kDa (lane 5) to only 61 kDa (lane 6), suggesting that some extension of glycans occurred in the ER. Part of the glycan addition was on the N-glycan, because after chase with TM, the increase of apparent molecular weight was much less (lane 8), than in the absence of TM.
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To study whether the moderate glycan extension in the sec23-1 cells was due to Golgi glycosyltransferases recycling to the ER, and not de novo synthesized transferases, we repeated the experiment in sec21-1 cells to block recycling from the Golgi to the ER. The apparent molecular weight of Scw4p increased similarly in the absence (lane 10) and presence of TM (lane 12), indicating little or no N-glycan extension. When this experiment was repeated using the sec18-1 mutant, similar results as shown in lanes 9-12 were obtained (not shown, strain H1628). Parallel samples labelled and chased in the absence of TM and immunoprecipitated with antibody against pentahistidine were subjected to reimmunoprecipitation with antiserum against 1,6-mannose. Scw4p from control cells (lane 13) and sec23-1 cells (lane 14) were recognized by the antiserum indicating glycosylation by Och1p. By contrast, Swp4p from sec21-1 cells appeared not to be decorated with
1,6-mannose (lane 15). Thus, in the absence of COPI traffic the glycans of Scw4p were not extended to the same extent than under conditions where transferases were allowed to relocate to the ER and remain there together with the substrate protein. These data substantiate the notion that it was the recycling Och1p molecules, rather than de novo synthesized ones, which performed the decoration of N-glycans in the ER.
Relocation of Och1p to the ER in the absence of COPII function
Finally, we wanted to verify morphologically that Och1p accumulates in the ER when ER exit of proteins is blocked. An Och1p version tagged with the hemagglutinin epitope (Och1p-HA) (Harris and Waters, 1996) was expressed under its own promoter in a sec23-1 mutant. Cells were incubated at 24°C in the presence of CHX in order to stop the synthesis of new proteins, and thereafter at 37°C to block ER exit. Then, cells were fixed and subjected to indirect immunofluorescent staining using antibody against the HA epitope. Mostly an ER-like staining was observed (Fig. 6A). Nomarski optics revealed the vacuole (Fig. 6B). Next we constructed a strain which co-expressed Och1p-HA and opsin-tagged mammalian cytochrome b(5), which is an ER-resident protein both in mammalian and S. cerevisiae cells (Yabal et al., 2003
). In double staining experiments HA polyclonal antibody (Fig. 6C) and opsin monoclonal antibody (Fig. 6D) stained similar structures (arrows). A similar HA antibody-stained cell sample as in Fig. 6A (Fig. 6E) was co-stained with DAPI (Fig. 6F) to reveal the nucleus. For the above experiments we performed the following controls using DAPI as nuclear marker. After growth of sec23-1 cells at 24°C, mostly dots, plus some nuclear membrane-like staining was detected (Fig. 7A), suggesting Golgi plus some ER localization. After chase at permissive temperature in the presence of CHX mostly dots were observed, suggesting that most of Och1p-HA was in the Golgi (Fig. 7B). Similar staining was observed when Och1p-HA was stained in a sec-7-1 mutant, where at 37°C membrane traffic is blocked in the Golgi (Fig. 7C). In normal cells Golgi-like staining was obtained for Och1p-HA at 24°C (Fig. 7D). These data support the conclusion based on our biochemical data above, that Och1p normally recycles between the Golgi and the ER. It is able to decorate protein-bound N-glycans in the ER, once it has access to the substrate glycoprotein for a sufficiently long time, which is the case when membrane traffic from the ER is blocked by mutations in COPII components.
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Discussion |
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Here we could extend the finding of retrograde transport in yeast cells of Golgi enzymes from N-glycan-specific transferases to transferases elongating O-glycans. We found that the O-glycans of the reporter protein Hsp150-ß-lactamase, blocked in the ER in COPII mutants, were extended. In normal cells the Hsp150
fragment is O-glycosylated at nearly all of its almost 70 serine and threonine residues with di-, tri-, tetra- and pentamannosides, occurring in the ratio of 4:1:1:1 (Jämsä et al., 1995
; Suntio et al., 1999
), whereas the ß-lactamase portion is not detectably glycosylated (Holkeri et al., 1996
). Lack of the 4th and 5th residues of the O-glycans of Hsp150
-ß-lactamase would correspond to a decrease of about 7 kDa (calculated molecular mass). However, subunit I remains attached to the fusion protein in the ER, adding a molecular mass of 9.7-13.5 kDa (determined by mass spectrometry) (Suntio et al., 1999
). Since ER-retained Hsp150
-ß-lactamase and the secreted fully glycosylated variant lacking subunit I co-migrated in SDS-PAGE, we suggest that the O-glycans of ER-retained Hsp150
-ß-lactamase were extended up to tri-mannosides. Such extensions are normally accomplished by Mnt1p, which is suggested to reside in the medial Golgi (Lussier et al., 1995b).
The recycling of Golgi transferases, rather than de novo synthesized transferases en route to the Golgi, appeared to be responsible for the Golgi-specific glycan extensions in the ER. This is based on the finding that the extensions could be diminished by two ways, by accumulation of the substrate protein in ER-derived vesicles and the transferases in Golgi-derived vesicles by blocking all vesicle fusion events with target membranes in an NSF-deficient sec18-1 mutant, and by abolishing specifically Golgi-to-ER traffic in a COPI-defective sec21-1 mutant, under which conditions also ER-to-Golgi traffic is blocked.
In summary, we found that the glycosyltransferase Och1p, responsible of starting the extension of the primary N-glycans in the Golgi by addition of an 1,6-mannose residue, recycles between the Golgi and the ER, and not only between the early and late Golgi as is currently thought. Moreover, Och1p was found to be functional in the ER, as shown by decoration by
1,6-mannose of several ER-blocked glycoproteins. Primary O-glycans were also extended in the ER, apparently by Mnt1p of the medial Golgi. By contrast, the N-glycans of ER-blocked invertase were not extended beyond the
1,6-mannose residue (Kaiser and Scheckman, 1990
). Nor appeared Mnn1p, adding the 4th and 5th mannose residues on O-glycans in the medial/trans Golgi, to elongate O-glycans of our ER-blocked reporter glycoprotein. Perhaps only the glycosyltransferases of early Golgi subcompartments recycle between the Golgi and the ER.
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Acknowledgments |
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References |
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