Glycobiology, 2002, Vol. 12, No. 11 30G-33G
© 2002 Oxford University Press
GLYCO-FORUM SECTION |
Letter to the Glyco-Forum
Hypoglycosylation in the alg12
yeast mutant destabilizes protease A and causes proteolytic loss of external invertase
3 Department of Biomedical Sciences, State University of New York at Albany School of Public Health, Albany, NY 12201, USA and 4 Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, NY 12201, USA
Received on April 12, 2002; revised on June 19, 2002; accepted on July 5, 2002
Abstract
The Saccharomyces cerevisiae alg12
mutant accumulates oligosaccharide lipid with a Man7GlcNAc2 oligosaccharide. To determine the N-glycan structures present on S. cerevisiae glycoproteins in the alg12 strain, we made attempts to purify external invertase, a highly glycosylated secreted protein. These efforts revealed that, in the alg12 background, external invertase was mildly hypoglycosylated and rapidly destroyed proteolytically. Although secreted alg9 invertase was more severely hypoglycosylated than the alg12 form, it was paradoxically stable during purification. The loss of periplasmic invertase was prevented by addition of pepstatin A to the cell cultures, suggesting that aspartyl proteases were active. We found that during overexpression of invertase in alg12 yeast, sufficient protease A was mistargeted to the periplasmic space, where it hydrolyzed the invertase. Even though alg9 invertase is underglycosylated in comparison to the alg12 form, it is more stable because in this genetic background much less protease A is secreted compared to alg12 cells. These observations may be relevant to studies using other extracellular proteins (e.g., mating factors, 
-glucosidase) as probes when characterizing glycosylation defects in yeast.
Key words: alg12 mutant/glycoprotein instability/invertase/protease A/S. cerevisiae
Introduction
Recent studies that focused on determining the N-glycan structures on alg12 yeast glycoproteins and on assessing the role played by individual sugars sequentially added in the synthesis of Glc3Man9GlcNAc2-PP-Dol (oligosaccharide lipid; OSL) in downstream glycan processing events (Cipollo and Trimble, 2002
) were hampered by extremely low yields of external invertase encoded by the SUC2 gene under the direction of the multicopy plasmid pRB58 (Carlson and Botstein, 1982) following derepression. Invertase is a useful N-glycan source because it is easily overexpressed, represents an end product of the secretory pathway, and is highly glycosylated, with an average of ~10 chains/60-kDa subunit (Ziegler et al., 1988), many of which are biosynthetic intermediates whose processing is terminated by protein folding (Trimble et al., 1983) and rapid compartmental transfer during secretion (Franzusoff, 1992). Wild-type yeast external invertase is generally stable in the periplasmic space and is quite resistant to proteolysis in cell extracts made for enzyme purification. In contrast, the nonglycosylated internal form of invertase is extremely sensitive to proteolysis (Williams et al., 1985).
In vivo, failure to add the upper-arm -1,6-linked mannose by Alg12p to OSL results in the retention of the middle-arm 1,2-Man cap during endoplasmic reticulum (ER) processing of the transferred glycan (Cipollo and Trimble, 2002), as predicted by in vitro studies on purified Mns1p (Ziegler and Trimble, 1991). Retention of this Man residue impairs outer chain mannan formation initiation by Och1P (Nakayama et al., 1997), leading to mild hypoglycosylation of external invertase. However, this is sufficient to markedly increase invertase’s sensitivity to vacuolar protease A (PrA), whose mistargeting to the periplasmic space is enhanced during overexpression of secreted invertase in the alg12 background.
Identification of external invertase proteolysis
Because Lussier and co-workers (1997) independently isolated alg12 (ecm39) as a potential cell wall mutant, it seemed possible that external invertase activities, measured by the method of Chu et al. (1978) following derepression of alg12 cells, were low because enzyme was leaking into the growth medium (appendix A in Burke et al., 2000) due to a weakened cell wall. As a positive control, we measured invertase activity in the medium of gda1 yeast, which are deficient in the Golgi GDPase required to form GMP from GDP. Because GMP is the antiporter for GDP-Man (Berninsone et al., 1994), the gda1 mutation prevents mannan elongation resulting in a leaky cell wall phenotype. Invertase was released into the gda1 growth medium on derepression, but not into the medium of alg9 or alg12 yeast cultures (data not shown), effectively ruling out cell wall defects as a loss of periplasmic invertase.
Cell-free extracts of alg9, alg12, and wild-type overexpressors of invertase were examined for vacuolar protease activities (Jones, 1990). Levels of carboxypeptidase Y (CPY) (1.4–1.7 U/mg) and protease B (PrB) (17–23 mU/mg) were similar in all three strains, but PrA levels varied over a nearly twofold range among the three strains. Table I shows that PrA levels were highest in wild-type cells (8.16 U/mg), intermediate in alg12 yeast (6.85 U/mg), and lowest in the alg9 strain (4.66 U/mg). Activity of PrA secreted into the growth media by spheroplasts prepared from the three strains in the presence of osmotic support (Burke et al., 2000) was assayed as a measure of periplasmically localized PrA. The range of secreted PrA from spheroplasts was similar in trend to that in the respective cell extracts but even more differentially distributed across the three strains, with the level being highest in wild-type (47.6 U/mg), intermediate in alg12 (21.9 U/mg), and lowest in alg9 cells (8.3 U/mg) (Table I).
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As seen in Figure 1A, alg9
and alg12 yeast produced two forms of PrA, one with both N-glycosylation sites occupied and another with only one site occupied. Endo H treatment of alg9, alg12, and wild-type PrA reduced all forms to an apparent Mr of 38 kDa (Figure 1A; compare lanes 2, 4, and 6), confirming that the difference in PrA molecular mass(es) among the three strains was due to their glycosylation states. Figure 1B (lanes 1 and 2) shows that the glycosylation pattern seen for PrA secreted by wild-type and alg12 spheroplasts was the same as that seen in a whole-cell extract (compare lanes 1 and 3 in Figure 1A). The level of PrA secreted by alg9 spheroplasts was too low for visualization by western blot analysis (Figure 1B, lane 3) but was measurable by enzyme assay (Table I). Figure 1C shows that both wild-type and alg12 PrA from spheroplasts collapsed to the same Mr on endo H treatment as did the PrA of whole-cell extracts from the two strains, indicating the identity of the PrA in these genetic backgrounds.
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To determine whether PrA present in the periplasmic space was the cause of invertase loss, we monitored external invertase production in wild-type (Figure 1D), alg9
(Figure 1E), and alg12 (Figure 1F) cells during glucose derepression over 5 h in the presence and absence of the aspartyl protease inhibitor pepstatin A. In the presence of pepstatin A, invertase from alg12 cells was stabilized to a greater extent than that secreted from either the alg9 or wild-type strains (compare panel F with panels D and E in Figure 1). These observations allowed large-scale purification of external invertase (Verostek et al., 1993) from alg12 cells for glycan structural studies (Cipollo and Trimble, 2002) by monitoring the level of invertase and PrA after derepression and adding 3 µM pepstatin A to the cultures and harvesting cells when the extracellular proteolytic activity began to increase. The wild type, alg12, and alg9 purified external invertases were compared by western blot analysis (Trimble et al., 1991; Verostek et al., 1993). Surprisingly, Figure 2 shows that alg12 invertase is less severely hypoglycosylated than was the alg9 preparation. This is likely due to the fact that N-glycans in alg12 invertase have one or two more Man residues in their core region and slightly longer outer chains than on the alg9 N-glycans, because truncation of mannose addition in the OSL synthesis pathway occurs one step later in the alg12 yeast mutant (Burda and Aebi, 1999).
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These data lead to a simple explanation that accounts for all of the observations regarding invertase loss in alg12
yeast. Overexpressors of invertase secrete vacuolar PrA into the periplasmic space. In wild-type cells, highly glycosylated secreted invertase is stable in the presence of high levels of periplasmic PrA activity. Although periplasmic PrA activity is somewhat lower in the alg12 genetic background, hypoglycosylated secreted invertase is degraded in the absence of protease inhibitors (Figure 1). Even though alg9 invertase is more severely underglycosylated than that from alg12 cells (Figure 2), it is not degraded due to the much-reduced periplasmic PrA activity in the alg9 genetic background (Table I).
Selective loss of PrA in alg9 and alg12 strains
Because CPY and PrB levels were similar among the three yeast strains, as noted, PrA levels appear to be selectively altered in the alg9 and alg12 strains. It is well documented that glycosylation protects some cellular proteins from proteolysis (Chu et al., 1978; Williams et al., 1985; O'Connor et al., 1996), and PrA was found to be underglycosylated in alg9 and alg12 strains (Figure 1). Thus, because PrA’s normal site of residence is the yeast vacuole, a site of high hydrolytic activity, glycosylation may ensure the protease’s stability, as in the case of invertase.
An alternative cause of underglycosylated PrA’s loss may relate to its folding and quality control editing in the ER. Alg9 and alg12 mutants fail to form and present the proper Man8GlcNAc2 structure to the protein folding/editing machinery shown to be involved in monitoring and eliminating misfolded proteins (Jakob et al., 1998). Kre6p, a protein required for ß1,6-glucan synthesis, also is selectively unstable in the gls1/cwh41 genetic background, a genotype that similarly fails to generate Man8GlcNAc2 in the ER (Abeijon et al., 1998).
PrA undergoes complex protein folding, which is dependent on its propeptide. Covalent linkage of the propeptide to the nascent protein is not required, as coexpression of the two portions is adequate to rescue proper folding of PrA (van den Hazel et al., 1993), thus implying that the propeptide acts as a PrA-specific chaperone. Recombinant forms of PrA lacking portions of the peopeptide (Klionsky et al., 1988) or the propeptide cleavage region (Finger et al., 1993) have been shown to be unstable and accumulate in the ER. Therefore, the decrease in PrA seen here and the instability of Kre6p in gls1/cwh41 noted may be intimately related to impaired protein editing that may occur in genetic backgrounds where characteristically truncated oligosaccharide structures are initially transferred to proteins from OSL.
Acknowledgments
The authors are grateful to Stephan te Heesen for supplying the alg9 and alg12 S. cerevisiae strains and for discussing results prior to publication. We are also grateful to Claudia Abeijon for supplying the gda1 S. cerevisiae strain. We thank Jakob R. Winther for supplying the Pep4p (PrA) antibody and for the insightful discussions concerning this protease. Preparation of this communication by Tracy Godfrey is deeply appreciated. This work was supported in part by U.S. Public Health Service grant GM23900 to R.B.T.
Abbreviations
CPY, carboxypeptidase Y; ER, endoplasmic reticulum; OSL, oligosaccharide lipid; PrA, protease A; PrB, protease B.
Footnotes
1 Present address: Boston University Goldman School of Dental Medicine, Boston, MA 02118–2392, USA 
2 To whom correspondence should be addressed; E-mail: trimble@wadsworth.org
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