Letter to the Glyco-Forum

Hypoglycosylation in the alg12{Delta} yeast mutant destabilizes protease A and causes proteolytic loss of external invertase

John F. Cipollo1,3 and Robert B. Trimble2,3,4

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{Delta} mutant accumulates oligosaccharide lipid with a Man7GlcNAc2 oligosaccharide. To determine the N-glycan structures present on S. cerevisiae glycoproteins in the alg12{Delta} strain, we made attempts to purify external invertase, a highly glycosylated secreted protein. These efforts revealed that, in the alg12{Delta} background, external invertase was mildly hypoglycosylated and rapidly destroyed proteolytically. Although secreted alg9{Delta} invertase was more severely hypoglycosylated than the alg12{Delta} 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{Delta} yeast, sufficient protease A was mistargeted to the periplasmic space, where it hydrolyzed the invertase. Even though alg9{Delta} invertase is underglycosylated in comparison to the alg12{Delta} form, it is more stable because in this genetic background much less protease A is secreted compared to alg12{Delta} cells. These observations may be relevant to studies using other extracellular proteins (e.g., mating factors, {alpha}-glucosidase) as probes when characterizing glycosylation defects in yeast.

Key words: alg12{Delta} mutant/glycoprotein instability/invertase/protease A/S. cerevisiae

Introduction

Recent studies that focused on determining the N-glycan structures on alg12{Delta} 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, 2002Go) 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, 1982Go) 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., 1988Go), many of which are biosynthetic intermediates whose processing is terminated by protein folding (Trimble et al., 1983Go) and rapid compartmental transfer during secretion (Franzusoff, 1992Go). 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., 1985Go).

In vivo, failure to add the upper-arm {alpha}-1,6-linked mannose by Alg12p to OSL results in the retention of the middle-arm {alpha}1,2-Man cap during endoplasmic reticulum (ER) processing of the transferred glycan (Cipollo and Trimble, 2002Go), as predicted by in vitro studies on purified Mns1p (Ziegler and Trimble, 1991Go). Retention of this Man residue impairs outer chain mannan formation initiation by Och1P (Nakayama et al., 1997Go), 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{Delta} background.

Identification of external invertase proteolysis

Because Lussier and co-workers (1997) independently isolated alg12{Delta} (ecm39) as a potential cell wall mutant, it seemed possible that external invertase activities, measured by the method of Chu et al. (1978)Go following derepression of alg12{Delta} cells, were low because enzyme was leaking into the growth medium (appendix A in Burke et al., 2000Go) due to a weakened cell wall. As a positive control, we measured invertase activity in the medium of gda1{Delta} 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., 1994Go), the gda1{Delta} mutation prevents mannan elongation resulting in a leaky cell wall phenotype. Invertase was released into the gda1{Delta} growth medium on derepression, but not into the medium of alg9{Delta} or alg12{Delta} yeast cultures (data not shown), effectively ruling out cell wall defects as a loss of periplasmic invertase.

Cell-free extracts of alg9{Delta}, alg12{Delta}, and wild-type overexpressors of invertase were examined for vacuolar protease activities (Jones, 1990Go). 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{Delta} yeast (6.85 U/mg), and lowest in the alg9{Delta} 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., 2000Go) 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{Delta} (21.9 U/mg), and lowest in alg9{Delta} cells (8.3 U/mg) (Table I).


View this table:
[in this window]
[in a new window]
 
Table I. Comparison of PrA activities in disrupted yeast cells with activities secreted by their spheroplasts
 
As seen in Figure 1A, alg9{Delta} and alg12{Delta} 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{Delta}, alg12{Delta}, 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{Delta} 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{Delta} 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{Delta} 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.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1. SUC2 overexpressors secrete PrA into the periplasmic space, where underglycosylated invertase is degraded. (A) Western blot of PrA in whole-cell extracts. Lanes 1, 3, and 5 are wild-type, alg12{Delta}, and alg9{Delta} PrA, respectively. Lanes 2, 4, and 6 are wild-type, alg12{Delta}, and alg9{Delta} PrA after digestion with endo H, respectively. (B) Western blot of PrA secreted by spheroplasts of wild-type (lane 1), alg12{Delta} (lane 2), and alg9{Delta} (lane 3) cells. (C) Western blot of spheroplast-secreted PrA of wild-type (lane 1) and alg12{Delta} (lane 3), and PrA of wild-type (lane 2) and alg12{Delta} (lane 4) after digestion with endo H. (DF) analysis of periplasmic invertase activity during glucose derepression in wild-type (D), alg9{Delta} (E), and alg12{Delta} (F) cells in the absence (X) and presence (•) of 5 µM pepstatin A. Cells (100-ml cultures) were derepressed by resuspension in SC medium (appendix A, Burke et al., 2000Go) + 0.1% Glc, and 5-ml aliquots were removed to tubes containing protease inhibitors (Williams et al., 1985Go) at the times indicated. Cells were pelleted, washed, and counted, and the invertase activity was measured (Chu et al., 1978Go).

 
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{Delta} (Figure 1E), and alg12{Delta} (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{Delta} cells was stabilized to a greater extent than that secreted from either the alg9{Delta} 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., 1993Go) from alg12{Delta} cells for glycan structural studies (Cipollo and Trimble, 2002Go) 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{Delta}, and alg9{Delta} purified external invertases were compared by western blot analysis (Trimble et al., 1991Go; Verostek et al., 1993Go). Surprisingly, Figure 2 shows that alg12{Delta} invertase is less severely hypoglycosylated than was the alg9{Delta} preparation. This is likely due to the fact that N-glycans in alg12{Delta} invertase have one or two more Man residues in their core region and slightly longer outer chains than on the alg9{Delta} N-glycans, because truncation of mannose addition in the OSL synthesis pathway occurs one step later in the alg12{Delta} yeast mutant (Burda and Aebi, 1999Go).



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2. Secreted invertase of alg12{Delta} is mildly hypoglycosylated. Western blots of wild-type (lane 1), alg12{Delta} (lane 2), and alg9{Delta} (lane 3) external invertases separated on a 4–20% precast SDS gel (Gradiapore).

 
These data lead to a simple explanation that accounts for all of the observations regarding invertase loss in alg12{Delta} 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{Delta} genetic background, hypoglycosylated secreted invertase is degraded in the absence of protease inhibitors (Figure 1). Even though alg9{Delta} invertase is more severely underglycosylated than that from alg12{Delta} cells (Figure 2), it is not degraded due to the much-reduced periplasmic PrA activity in the alg9{Delta} genetic background (Table I).

Selective loss of PrA in alg9{Delta} and alg12{Delta} 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{Delta} and alg12{Delta} strains. It is well documented that glycosylation protects some cellular proteins from proteolysis (Chu et al., 1978Go; Williams et al., 1985Go; O'Connor et al., 1996Go), and PrA was found to be underglycosylated in alg9{Delta} and alg12{Delta} 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{Delta} and alg12{Delta} 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., 1998Go). Kre6p, a protein required for ß1,6-glucan synthesis, also is selectively unstable in the gls1/cwh41{Delta} genetic background, a genotype that similarly fails to generate Man8GlcNAc2 in the ER (Abeijon et al., 1998Go).

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., 1993Go), thus implying that the propeptide acts as a PrA-specific chaperone. Recombinant forms of PrA lacking portions of the peopeptide (Klionsky et al., 1988Go) or the propeptide cleavage region (Finger et al., 1993Go) 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{Delta} 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{Delta} and alg12{Delta} S. cerevisiae strains and for discussing results prior to publication. We are also grateful to Claudia Abeijon for supplying the gda1{Delta} 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 Back

2 To whom correspondence should be addressed; E-mail: trimble@wadsworth.org Back

References

Abeijon, C. and Chen, L.Y. (1998) The role of glucosidase I (Cwh41p) in the biosynthesis of cell wall beta-1, 6-glucan is indirect. Mol. Biol. Cell, 9, 2729–2738.[Abstract/Free Full Text]

Berninsone, P., Miret, J.J., and Hirschberg, C.B. (1994) The Golgi guanosine diphosphatase is required for transport of GDP-mannose into the lumen of Saccharomyces cerevisiae Golgi vesicles. J. Biol. Chem., 269, 207–211.[Abstract/Free Full Text]

Burda, P. and Aebi, M. (1999) The dolichol pathway of N-linked glycosylation. Biochim. Biophys. Acta, 1426, 239–257.[ISI][Medline]

Burke, D., Dawson, D., and Stearns, T. (2000) Methods in yeast genetics.

Carlson, M. and Botstein, D. (1982) Two differentially regulated mRNAs with different 5' ends encode secreted and intracellular forms of yeast invertase. Cell, 28, 145–154.[ISI][Medline]

Chu, F.K., Trimble, R.B., and Maley, F. (1978) The effect of carbohydrate depletion on the properties of yeast external invertase. J. Biol. Chem., 253, 8691–8693.[ISI][Medline]

Cipollo, J.F. and Trimble, R.B. (2002) The Saccharomyces cerevisiae alg12{Delta} mutant reveals a role for the middle-arm {alpha}1, 2Man- and upper-arm {alpha}1, 2Man{alpha}1,6Man- residues of Glc3Man9GlcNAc2-PP-Dol in regulating glycoprotein glycan processing in the endoplasmic reticulum and Golgi apparatus. Glycobiology, 12 (in press).

Finger, A., Knop, M., and Wolf, D.H. (1993) Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast. Eur. J. Biochem., 218, 565–574.[Abstract]

Franzusoff, A. (1992) Beauty and the yeast: compartmental organization of the yeast secretory pathway. Semin. Cell Biol., 3, 309–324.[Medline]

Jakob, C.A., Burda, P., Roth, J., and Aebi, M. (1998) Degradation of misfolded endoplsamic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure. J. Cell Biol., 142, 1223–1233.[Abstract/Free Full Text]

Jones, E.W. (1990) Vacuolar proteases in yeast Saccharomyces cerevisiae. Methods Enzymol., 185, 372–386.[Medline]

Klionsky, D.J., Banta, L.M., and Emr, S.D. (1988) Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information. Mol. Cell. Biol., 8, 2105–2116.[ISI][Medline]

Lussier, M., White, A.M., Sheraton, J., di Paolo, T., Treadwell, J., Southard, S.B., Horenstein, C.I., Chen-Weiner, J., Ram, A.F., Kapteyn, J.C., and others. (1997) Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics, 147, 435–450.[Abstract/Free Full Text]

Nakayama, K., Nokanistu-Shindo, Y., Tanaka, A, Haga-Toda, Y., and Jigami, Y. (1997) Substrate specificity of alpha-1, 6-mannosyltransferase that initiates N-linked mannose outer chain elongation in Saccharomyces cerevisiae. FEBS Lett., 412, 547–550.[CrossRef][ISI][Medline]

O’Connor, S.E. and Imperiali, B. (1996) Modulation of protein structure and function by asparagine-linked glycosylation. Chem. Biol., 3, 803–812.[ISI][Medline]

Trimble, R.B., Atkinson, P.H., Tschopp, J.F., Townsend, R.R., and Maley, F. (1991) Structure of the oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichic pastoris. J. Biol. Chem., 266, 22807–22817.[Abstract/Free Full Text]

Trimble, R.B., Maley, F., and Chu, F.K. (1983) Glycoprotein biosynthesis in yeast. Protein confirmation affects processing of high mannose oligosaccharides on carboxypeptidase Y and invertase. J. Biol. Chem., 258, 2562–2567.[Free Full Text]

van den Hazel, H.B., Kielland-Brandt, M.C., and Winther, J.R. (1993) The propeptide is required for in vitro formation of stable active yeast proteinase A and can function even when not covalently linked to the mature region. J. Biol. Chem., 268, 18002–18007.[Abstract/Free Full Text]

Verostek, M.F., Atkinson, P.A., and Trimble, R.B. (1993) Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. I. Role of glucose in the initial glycosylation of invertase in the endoplasmic reticulum. J. Biol. Chem., 268, 12095–12103.[Abstract/Free Full Text]

Williams, R.S., Trumbly, R.J., MacColl, R., Trimble, R.B., and Maley F. (1985) Comparative properties of amplified external and internal invertase from the yeast SUC2 gene. J. Biol. Chem., 260, 13334–13341.[Abstract/Free Full Text]

Ziegler, F.D. Trimble, R.B. (1991) Glycoprotein biosynthesis in yeast: purification and characterization of the endoplasmic reticulum Man9 processing {alpha}-mannosidase. Glycobiology, 1, 605–614.[Abstract]

Ziegler, F.D., Maley, F., and Trimble, R.B. (1988) Characterization of the glycosylation sites in yeast external invertase. II. Location of the endo-{alpha}-N-acetylglucosaminidase H-resistant sequons. J. Biol. Chem., 263, 6986–6992.[Abstract/Free Full Text]