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Mutants of Kluyveromyces lactis with altered protein glycosylation are affected in cell wall morphogenesis
Res. Microbiol. 150 (1999) 5−12 © Elsevier, Paris
Mutants of Kluyveromyces lactis with altered protein glycosylation are affected in cell wall morphogenesis Daniela Uccelletti, Francesca Farina, Alessandro Morlupi, Claudio Palleschi* Foundation Institut Pasteur-Fondazione Cenci-Bolognetti, Department of Developmental and Cell Biology, University of Rome ’La Sapienza’, Piazza Aldo Moro 5, 00185 Rome, Italy (Submitted 28 July 1998; accepted 2 October 1998)
Abstract — We isolated spontaneous mutants resistant to sodium orthovanadate in the biotechnologically significant yeast Kluyveromyces lactis. Resistance behaved as a recessive character in all mutants analyzed. Four genes were defined by complementation analysis, from vga1 to vga4. These mutants showed defects in N-linked as well as O-linked glycosylation processes. In addition, the mutants exhibited sensitivity to the aminoglycoside hygromycin B and to calcofluor white, with the exception of vga4; this mutant grew in the presence of the antibiotic as well as the parental wild type and was resistant to calcofluor. The mutations were accompanied by alterations in the cell wall structure, as revealed by the delocalisation of chitin, changes in cell shape and size and by the clumpy aspect of the cultures. The mutants isolated provide basic tools for molecular and cellular analysis of glycosylation processes in K. lactis. © Elsevier, Paris Kluyveromyces lactis / glycosylation / vanadate / chitin / cell wall morphogenesis / mutant
1. Introduction Kluyveromyces lactis is a biotechnologically significant yeast and it is already being exploited as a host for the production of heterologous proteins due to its secretory performances [6, 13, 26, 28, 29]. Nevertheless, the secretion and glycosylation processes in Kluyveromyces yeasts are at present virtually unexplored. The biogenesis of cell wall mannoproteins has been investigated to some extent in K. lactis; three classes of mutants have been isolated by Ballou and co-workers, and are defective in specific steps of mannoprotein biosynthesis [10, 11, 24]. One of the mutants,
* Correspondence and reprints Tel.: 39 06 499 12 321; fax: 39 6 499 12 351; [email protected] Abbreviations: ER, endoplasmic reticulum; HygB, hygromycin B; ME, malt extract; NaV, sodium orthovanadate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulphate/polyacrylamide gel electrophoresis; UDPGlcNAc, uridine diphosphate N-acetylglucosamine.
mnn2-2, has been extensively characterised [1] and the corresponding gene encodes for the UDP-GlcNAc transporter of the Golgi apparatus [2]. The study of the K. lactis genes controlling the glycosylation processes could thus offer relevant insights into the secretion process and could possibly be of practical impact in the production of heterologous proteins. Within the framework of a project aimed at analysing the basis of K. lactis secretory capabilities, we focused our attention on genes controlling the processing of glycoproteins. To this end, we isolated glycosylation mutants of K. lactis, taking advantage of the knowledge that, in S. cerevisiae, the orthovanadate-resistant mutants usually show defects in protein glycosylation [4, 15]. Orthovanadate is a biologically significant form of vanadium and it has been suggested to share structural similarity with orthophosphate [5, 17]. Orthovanadate resistance has been analysed in S. cerevisiae [18, 31, 32] and has also been found to be involved in protein phosphorylation and growth control [14]. We report here
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the isolation and characterization of K. lactis mutants resistant to orthovanadate and defective in the glycosylation pathway.
2. Materials and methods 2.1. Yeast strains and media
The K. lactis strains used were MW278-20C (MATα, ade2-1, leu2, uraA1-1, lac4-8) and CBS 2359/152 (MATa, metA). Complete medium (YPD), minimal medium and sporulation medium (ME) were prepared as reported by Wesolowsky-Louvel et al. [30]. A stock solution of 1 M sodium orthovanadate (Sigma) was prepared in water, filter-sterilized and freshly used on YPD plates to a final concentration of 7 mM. The calcofluor white resistance test was performed by adding to YPD plates calcofluor white (Sigma) at different concentrations from a stock solution of 1 mg/mL, filter-sterilized. 2.2. Isolation of mutants
Mutants were isolated from stationary phase cells of strains MW278-20C and CBS 2359/152, grown in YPD medium at 30 °C and plated at 108 cells/plate onto YPD containing 7 mM sodium orthovanadate. After 4 days, colonies were picked and restreaked five times on the same medium to confirm the homogeneity of the cultures and the consistency of the orthovanadate resistance (VanR) phenotype. 2.3. Complementation analysis
Complementation analysis was done by crossing on ME plates ten mutants derived from MW278-20C with each of the ten mutants of opposite mating type derived from CBS2359/152. The resulting 100 diploids were selected on minimal medium and then streaked to obtain single colonies. Cultures from single colonies were tested for growth on plates containing 7 mM vanadate and assayed for the glycosylation level of invertase. The diploid obtained by crossing MW278-20C with CBS 2359/152 strains was used as a control.
2.4. Analysis of invertase
Preparation of the invertase extracts, native PAGE of the invertase and activity staining were as described by Ballou [3] with the following minor modifications: the concentration of polyacrylamide in the gels was lowered to 3.5% and the 15-cm-long slab gels were run for 18 h at 15 mA constant current to better resolve the highly glycosylated forms of invertase. 2.5. Analysis of chitinase
Native chitinase was purified from stationary cultures of K. lactis grown in YPD by using the method described by Kuranda and Robbins [16] for S. cerevisiae and detected by Western blotting using rabbit IgG antibodies against deglycosylated chitinase (antibodies were a generous gift of Dr. Widmar Tanner, University of Regensburg, Germany). Final visualization was obtained with anti-rabbit IgG conjugated with peroxidase and the ECL detection kit (Amersham). 2.6. Fluorescence microscopy
Chitin staining was performed as follows: aliquots of 107 cells of overnight cultures in YPD were centrifuged and washed two times with PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 138 mM NaCl, 1.8 mM KCl). The cell pellets were then resuspended in 100 µL of 1 mg/mL calcofluor white (Sigma) and incubated for 10 min at room temperature. The cells were then washed three times with 1 mL of PBS and resuspended with 500 µL of distilled water [23]. Aliquots were applied to slides and observed with an UV filter in a fluorescence microscope.
3. Results 3.1. Characterisation of K. lactis vga mutants
In order to study the glycosylation processes of K. lactis, spontaneous orthovanadateresistant mutants were selected from two strains of opposite mating type (see Materials and methods). The mutants of both strains were
K. lactis mutants and glycosylation
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Table I. Yeast strains. Strain
Genotype
Source
MW278-20C CBS2359/152 CPV1 CPV2 CPV3 CPV4 CPV5 CPV6 CPV7 CPV8
MATα ade2-1 leu2 uraA1-1 lac4-8 MATα metA MATα ade2-1 leu2 uraA1-1 lac4-8 vga1 MATα ade2-1 leu2 uraA1-1 lac4-8 vga2 MATα ade2-1 leu2 uraA1-1 lac4-8 vga3 MATα ade2-1 leu2 uraA1-1 lac4-8 vga4 MATa metA vga1 MATa metA vga2 MATa metA vga3 MATa metA vga4
Wesolowsky-Louvel Wesolowsky-Louvel Spontaneous mutant from MW278-20C Spontaneous mutant from MW278-20C Spontaneous mutant from MW278-20C Spontaneous mutant from MW278-20C Spontaneous mutant from CBS2359/152 Spontaneous mutant from CBS2359/152 Spontaneous mutant from CBS2359/152 Spontaneous mutant from CBS2359/152
isolated on YPD plates containing 7 mM of sodium orthovanadate (NaV); the minimal inhibitory concentration of NaV for the wild type strains was 5 mM. The vanadate-resistant colonies from MW278-20C (MATα) and CBS 2359/152 (MATa) appeared at a frequency of approximately 10–7. Among them, ten mutants of each strain were randomly chosen to determine the recessiveness or dominance of the mutations. To this aim, we crossed the mutants derived from MW278-20C with the CBS 2359/152 wild type strain and vice versa for the mutants derived from CBS 2359/152. The diploids were then analysed for their capability to grow on rich medium containing 7 mM NaV; all the diploid strains were unable to grow after 4 days of incubation, indicating that the vanadate-resistance mutations tested were recessive. In order to verify whether the mutants isolated had a single mutation, the same diploids were sporulated on malt extract and the resulting asci dissected. Ten asci were typically analysed for each diploid. The segregation of phenotypes VanR:VanS was always 2:2; this is the expected behaviour of single mutations (data not shown). The mutants were mated together in a combinatory procedure to determine the number of complementation groups represented. The diploids were then assayed for vanadate resistance and glycosylation defects of invertase. The results of the complementation analysis are presented in figure 1. The mutations analysed were assigned to four complementation groups and
each group contained more than one isolate. The four groups were named vga1 to vga4 (for vanadate glycosylation affected, see next section) and a representative isogenic strain from each group (CPV1 to CPV4; see table I) was used therein for the characterisation of the mutations. We first analysed the level of vanadate resistance. As shown in table II, CPV1 was the most resistant, CPV2 and CPV3 grew well up to 8 mM NaV. Since the vanadate-resistant mutants of S. cerevisiae have often been reported to be hypersensitive to aminoglycosides [8], we
Figure 1. Native gel electrophoresis of the active invertase from vga mutants and complementation between vga mutants based on invertase mobility. Lane 1, MW278-20C, wild type; lane 2, CPV1 × CPV6 diploid; lane 3, CPV1 × CPV7 diploid; lane 4, CPV1 × CPV8 diploid; lane 5, CPV2 × CPV7 diploid; lane 6, CPV2 × CPV8 diploid; lane 7, CPV3 × CPV8 diploid; lane 8, CPV4 haploid; lane 9, CPV3 haploid; lane 10, CPV2 haploid; lane 11, CPV1 haploid; lane 12, CBS 2359/152, wild type.
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Table II. Phenotypic analysis of vanadate-resistant mutants. Strains MW278-20C CPV1 CPV2 CPV3 CPV4
NaV (mM)
HygB (µg/mL)
Thermosensitivity
Calcofluor (µg/mL)
7
10
1.5
2.5
5
36 °C
37 °C
38 °C
10
15
40
– +++ +++ +++ +++
– ++ +/– +/– +
+++ +/– ++ ++ +++
+++ +/– +/– + +++
+++ +/– – – +++
+++ +/– + – +++
+++ – – – +
+ – – – –
+++ +++ +++ – +++
+++ – – – +++
+/– – – – +++
Growth of the cells was scored as very strong (+++), strong (++), weak (+), papillae (+/–) or none (–). The thermosensitivity test was performed comparing the growth of the cell at the indicated temperatures with respect to growth at 24 °C.
tested the resistance to hygromycin B (HygB) of vga mutants. The wild type K. lactis strains we studied were not able to grow at HygB concentrations above 40 µg/mL (not shown). The results reported in table II show that the vanadate-resistant mutants of K. lactis were affected to a variable extent in the resistance to HygB. An exception was CPV4; the resistance to HygB of this mutant was indistinguishable from that of the parental strain; in fact, CPV4 was able to grow in the presence of 40 µg/mL of HygB. We then studied the temperature dependence of the growth of vga mutants in YPD medium. The wild type strain was still able to grow, albeit slowly, at 38 °C, whereas the mutants were inhibited. Moreover, the CPV1, CPV2 and CPV3 strains failed to grow at 36 °C, whereas the CPV4 mutant was able to grow at this temperature and did not grow at 38 °C (see table II). Mutations altering N-linked glycosylation (see next section) are expected to affect the cell wall mannoproteins travelling trough the secretory apparatus. This may result in alterations of the cell wall structure that can be revealed by changes in the resistance to calcofluor white. We analysed the growth of mutants in YPD supplemented with various concentrations of this dye. As shown in table II, we observed sensitivity to calcofluor for all but one vga mutant, with CPV3 being more sensitive with respect to the others. The CPV4 mutant was resistant up to 40 µg/mL of calcofluor, while the wild type grew well up to 30 µg/mL.
Taken all together, the results in table II show that the four complementation groups had distinctive phenotypic traits. 3.2. Analysis of glycosylation defects in vga mutants
We made use of invertase as a reporter protein in order to investigate the defects in the N-linked glycosylation pathway of our mutants since, in S. cerevisiae, this protein can only be N-glycosylated [25]. The selected strains CPV1 to CPV4, representative of each complementation group, were analysed for the production of active invertase by native gel electrophoresis. Wild type cells secreted a population of glycosylated invertase visible as a diffuse band (figure 1, lanes 1 and 12); this was due to the heterogeneous size of the mannose outer chains N-linked to the secreted invertase [3]. As shown in figure 1, lanes 9, 10 and 11, strains with the vga1, vga2 and vga3 mutations produced only a fast-migrating form of invertase, representing the core- or ER-glycosylated form of this enzyme [12]. The CPV4 strain secreted slightly underglycosylated forms of invertase relative to wild type cells (figure 1, lane 8). We also analysed the invertase secreted from diploids obtained by crossing the mutants in a combinatory scheme. The results reported in figure 1, lanes 2 to 7, show that each combination of the studied mutations resulted in the production of fully glycosylated invertase forms having the same electrophoretic mobility as those produced by the wild type strains.
K. lactis mutants and glycosylation
We observed at least two different defects in the electrophoretic profile of the secreted invertase; this indicated that different steps of the N-linked glycosylation pathway were impaired in the analysed mutants. We then analysed the O-linked glycosylation of our mutants, employing as a reporter the chitinase. This protein is secreted from yeast cells into the growth medium and is exclusively O-mannosylated [16, 20]. Chitinase can be purified from the culture supernatant by selective chitin binding. Subsequent analysis by SDSPAGE is based on the assumption that the electrophoretic mobility of chitinase depends on the amount of the O-linked mannose [16]. Results reported in figure 2 show that the chitinase secreted by the CPV4 cells had greater mobility than that of the wild type counterpart (compare lanes 2 and 3), suggesting that the amount of O-linked mannose is reduced in this strain. The CPV3 mutant instead produced a chitinase with mobility indistinguishable from that of the wild type parent (compare lanes 1 and 3). This indicates that O-linked glycosylation of the CPV3 cells is probably unaffected; on the other hand, this mutant is strongly impaired in the N-linked glycosylation pathway (see figure 1). The chitinase was not detected in the culture supernatant of CPV1 and CPV2 strains; this could be ascribed to defects in the secretory pathway and remains to be investigated. 3.3. The K. lactis vga mutants are impaired in the polarized deposition of chitin
Alterations in the glycosylation pathway are expected to affect the production and delivery of enzymes and glycoproteins devoted to the organisation of the cell wall. The chitin, a linear β-1,4-N-acetylglucosamine polymer, is a minor but highly structured component of the yeast cell wall; it is mostly found in the bud scar, the ring-like structure that marks the bud emergence site. The localized deposition of chitin results from a highly organized delivery of various proteins through the polarised secretion apparatus.
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Figure 2. Immunoblot analysis of chitinase secreted from isogenic vga mutants. Lane 1, CPV3; lane 2, CPV4; lane 3, MW278-20C, wild type. Molecular mass marker on the right, plain arrow.
We thus investigated the morphology of chitin organisation in our mutants using fluorescence microscopy of calcofluor white stained cells. The results are reported in figure 3. The wild type cells showed the typical localisation of chitin at the neck between mother cells and emerging buds and at the rings of the bud scars (figure 3, panel A). The cultures of CPV1, CPV2 and CPV3 mutants (figure 3, panels B, C and D, respectively) exhibited a clumpy aspect and remarkable heterogeneity in cell morphology. A vast majority of the cells were large and round; the so-called ’Mickey Mouse’ shape was also present. The larger cells had a bright fluorescence staining pattern uniformly distributed all around the cell wall; in the smaller cells, chitin accumulation at the actively growing sites was still visible, together with calcofluor stained material diffused all around the cell wall. All these observations indicate that the localized deposition of chitin is impaired in these mutants; this defect is often referred to as loss of polarized growth. The CPV4 mutant (figure 3, panel E) exhibited different morphological changes, with the chitin staining pattern and cell shape very similar to the wild type ones; the cell size of the mutant was, however, larger than normal. The changes in the cell shape and size we observed in all the mutants studied were not due to osmotic defects, since they were not affected by the addition of 1 M sorbitol to the medium (results not shown).
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Figure 3. Analysis of chitin localisation in the isogenic vga mutants by fluorescence microscopy. Cells were grown to late exponential phase in YPD and stained with calcofluor white. All the pictures were at the same magnification. A. MW278-20C, wild type. B. CPV1. C. CPV2. D. CPV3. E. CPV4.
4. Discussion The mechanism of resistance to sodium orthovanadate in yeast cells is not completely understood; however, S. cerevisiae-resistant mutants isolated by four laboratories [4, 14, 22, 32] demonstrate a strong correlation of the vanadate-resistant phenotype with alterations in the glycosylation processes. We thus isolated spontaneous orthovanadate-resistant mutants of K. lactis affected in the glycosylation pathway of this biotechnologically significant yeast. These mutants fall into at least four complementation groups, and all of them exhibit defects in the N-linked glycosylation pathway, as revealed
by the electrophoretic profiles of the secreted invertase. This approach, proposed by Ballou [3], can reveal alterations in the mannose outer chain elongation of the secreted proteins, and this modification is a landmark event of the Golgi apparatus [4]. One of our mutants is also impaired in O-linked glycosylation, as indicated by the electrophoretic mobility shift of chitinase [16]. The K. lactis mutants we isolated are therefore affected in various glycosylation steps that presumably occur in the early phases of the secretory process. Dean [8] reported that S. cerevisiae mutants defective in all aspects of glycosylation are
K. lactis mutants and glycosylation
specifically sensitive to aminoglycosides, and suggested that the degree of sensitivity is related to the severity of glycosylation abnormalities. Our analysis reveals that K. lactis mutants present different degrees of sensitivity to HygB, with the exception of the CPV4 mutant that has the same HygB resistance level as the wild type parent. On the other hand, the CPV4 strain has an intermediate glycosylation profile, similar to the vrg4 and alg4 mutants of S. cerevisiae [19, 22], but both vrg4 and alg4 mutants are hypersensitive to HygB. This trait of the CPV4 strain could possibly offer novel insights into the still unclear link between glycosylation defects and sensitivity to aminoglycosides. Changes in the glycosylation processes could affect the secretion and delivery machinery impinging upon the organisation of the outer cell structures. The involvement of the genes identified by the analysed mutations in the assembly of the cell wall is supported by the altered resistance to calcofluor white and by fluorescence microscopy observations. We found both calcofluor hypersensitive (CPV1, CPV2 and CPV3) as well as resistant phenotypes (CPV4). Hypersensitivity to calcofluor is associated with delocalisation of chitin all along the cell wall. The deposition of chitin at the sites of active growth results from the accurate localisation of chitin synthase activities [7] also in coordination with several other proteins such as the septins [9]. Mondesert and co-workers [19] recently reported that N-linked glycosylation processes, together with polarization of the secretory machinery, control polarized growth and cell wall morphogenesis in S. cerevisiae. Mutants with altered glycosylation described here suggest that a similar connection can be established in K. lactis, and confirm that the mechanism of resistance to orthovanadate involves the glycosylation pathway. The characteristics of the CPV4 mutant are somewhat unusual: this mutant has a chitin localisation indistinguishable from that of the wild type by fluorescence microscopy and is hyperresistant to calcofluor white. The rare calcofluor-resistant mutants of S. cerevisiae are usually characterised by a decrease in chitin in
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the cell wall and in the activity of chitin synthase III, as reported for cal2 [27] or dit101 mutants, where the chitin is almost undetectable [21]. Resistance to the toxic compound thus results from the reduction or from the absence of the target molecule on the cell surface. In the CPV4 mutant of K. lactis, a novel resistance mechanism could be present; we are tempted to speculate that the glycosylation changes occurring in this mutant could affect glycoprotein(s) present in the cell wall architecture that interact with chitin. The described mutants should represent basic tools for the molecular genetics and cell biology studies of the glycosylation processes in K. lactis. Résumé — Mutants de Kluyveromyces lactis, altération de la glycosylation protéinique et morphogenèse pariétale. Chez la levure intéressante en biotechnologie, Kluyveromyces lactis, nous avons isolé des mutants spontanés, résistants à l’orthovanate sodique. La résistance est du type récessif chez tous les mutants étudiés. Quatre gènes ont été définis par complémentation, de vga1 à vga4. Ces mutants sont défectifs pour la glycosylation liée à N aussi bien qu’à O. De plus, ils sont sensibles à l’hygromicine B, un aminoglycoside, et au calcofluor, à l’exception du mutant vga4 qui se développe en présence de l’antibiotique mais qui résiste au calcofluor. Les mutations entraînent des altérations de la structure pariétale, révélée par une délocalisation de la chitine, des modifications de la forme et de la taille cellulaires et un aspect compact des cultures. Les mutants isolés peuvent servir d’outils de base dans l’analyse moléculaire et cellulaire des processus de glycosylation chez K. lactis. © Elsevier, Paris glycosylation / morphogenèse de la paroi cellulaire / Kluyveromyces lactis / mutant / chitine / vanadate
Acknowledgments We are indebted to Dr. Wesolowsky-Louvel for K. lactis wild type strains and helpful advice, and to Dr. Tanner for antibodies. We also thank Mr. Castelli for excellent technical assistance. This work was supported by the CNR Target Project on Biotechnology no. 97.01162.49, by the Commission of the European Community
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(BIO4-CT96-0003) and by MURST-University La Sapienza Cofin 1997.
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Mutants of Kluyveromyces lactis with altered protein glycosylation are affected in cell wall morphogenesis Daniela Uccelletti, Francesca Farina, Alessandro Morlupi, Claudio Palleschi* Foundation Institut Pasteur-Fondazione Cenci-Bolognetti, Department of Developmental and Cell Biology, University of Rome ’La Sapienza’, Piazza Aldo Moro 5, 00185 Rome, Italy (Submitted 28 July 1998; accepted 2 October 1998)
Abstract — We isolated spontaneous mutants resistant to sodium orthovanadate in the biotechnologically significant yeast Kluyveromyces lactis. Resistance behaved as a recessive character in all mutants analyzed. Four genes were defined by complementation analysis, from vga1 to vga4. These mutants showed defects in N-linked as well as O-linked glycosylation processes. In addition, the mutants exhibited sensitivity to the aminoglycoside hygromycin B and to calcofluor white, with the exception of vga4; this mutant grew in the presence of the antibiotic as well as the parental wild type and was resistant to calcofluor. The mutations were accompanied by alterations in the cell wall structure, as revealed by the delocalisation of chitin, changes in cell shape and size and by the clumpy aspect of the cultures. The mutants isolated provide basic tools for molecular and cellular analysis of glycosylation processes in K. lactis. © Elsevier, Paris Kluyveromyces lactis / glycosylation / vanadate / chitin / cell wall morphogenesis / mutant
1. Introduction Kluyveromyces lactis is a biotechnologically significant yeast and it is already being exploited as a host for the production of heterologous proteins due to its secretory performances [6, 13, 26, 28, 29]. Nevertheless, the secretion and glycosylation processes in Kluyveromyces yeasts are at present virtually unexplored. The biogenesis of cell wall mannoproteins has been investigated to some extent in K. lactis; three classes of mutants have been isolated by Ballou and co-workers, and are defective in specific steps of mannoprotein biosynthesis [10, 11, 24]. One of the mutants,
* Correspondence and reprints Tel.: 39 06 499 12 321; fax: 39 6 499 12 351; [email protected] Abbreviations: ER, endoplasmic reticulum; HygB, hygromycin B; ME, malt extract; NaV, sodium orthovanadate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulphate/polyacrylamide gel electrophoresis; UDPGlcNAc, uridine diphosphate N-acetylglucosamine.
mnn2-2, has been extensively characterised [1] and the corresponding gene encodes for the UDP-GlcNAc transporter of the Golgi apparatus [2]. The study of the K. lactis genes controlling the glycosylation processes could thus offer relevant insights into the secretion process and could possibly be of practical impact in the production of heterologous proteins. Within the framework of a project aimed at analysing the basis of K. lactis secretory capabilities, we focused our attention on genes controlling the processing of glycoproteins. To this end, we isolated glycosylation mutants of K. lactis, taking advantage of the knowledge that, in S. cerevisiae, the orthovanadate-resistant mutants usually show defects in protein glycosylation [4, 15]. Orthovanadate is a biologically significant form of vanadium and it has been suggested to share structural similarity with orthophosphate [5, 17]. Orthovanadate resistance has been analysed in S. cerevisiae [18, 31, 32] and has also been found to be involved in protein phosphorylation and growth control [14]. We report here
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the isolation and characterization of K. lactis mutants resistant to orthovanadate and defective in the glycosylation pathway.
2. Materials and methods 2.1. Yeast strains and media
The K. lactis strains used were MW278-20C (MATα, ade2-1, leu2, uraA1-1, lac4-8) and CBS 2359/152 (MATa, metA). Complete medium (YPD), minimal medium and sporulation medium (ME) were prepared as reported by Wesolowsky-Louvel et al. [30]. A stock solution of 1 M sodium orthovanadate (Sigma) was prepared in water, filter-sterilized and freshly used on YPD plates to a final concentration of 7 mM. The calcofluor white resistance test was performed by adding to YPD plates calcofluor white (Sigma) at different concentrations from a stock solution of 1 mg/mL, filter-sterilized. 2.2. Isolation of mutants
Mutants were isolated from stationary phase cells of strains MW278-20C and CBS 2359/152, grown in YPD medium at 30 °C and plated at 108 cells/plate onto YPD containing 7 mM sodium orthovanadate. After 4 days, colonies were picked and restreaked five times on the same medium to confirm the homogeneity of the cultures and the consistency of the orthovanadate resistance (VanR) phenotype. 2.3. Complementation analysis
Complementation analysis was done by crossing on ME plates ten mutants derived from MW278-20C with each of the ten mutants of opposite mating type derived from CBS2359/152. The resulting 100 diploids were selected on minimal medium and then streaked to obtain single colonies. Cultures from single colonies were tested for growth on plates containing 7 mM vanadate and assayed for the glycosylation level of invertase. The diploid obtained by crossing MW278-20C with CBS 2359/152 strains was used as a control.
2.4. Analysis of invertase
Preparation of the invertase extracts, native PAGE of the invertase and activity staining were as described by Ballou [3] with the following minor modifications: the concentration of polyacrylamide in the gels was lowered to 3.5% and the 15-cm-long slab gels were run for 18 h at 15 mA constant current to better resolve the highly glycosylated forms of invertase. 2.5. Analysis of chitinase
Native chitinase was purified from stationary cultures of K. lactis grown in YPD by using the method described by Kuranda and Robbins [16] for S. cerevisiae and detected by Western blotting using rabbit IgG antibodies against deglycosylated chitinase (antibodies were a generous gift of Dr. Widmar Tanner, University of Regensburg, Germany). Final visualization was obtained with anti-rabbit IgG conjugated with peroxidase and the ECL detection kit (Amersham). 2.6. Fluorescence microscopy
Chitin staining was performed as follows: aliquots of 107 cells of overnight cultures in YPD were centrifuged and washed two times with PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 138 mM NaCl, 1.8 mM KCl). The cell pellets were then resuspended in 100 µL of 1 mg/mL calcofluor white (Sigma) and incubated for 10 min at room temperature. The cells were then washed three times with 1 mL of PBS and resuspended with 500 µL of distilled water [23]. Aliquots were applied to slides and observed with an UV filter in a fluorescence microscope.
3. Results 3.1. Characterisation of K. lactis vga mutants
In order to study the glycosylation processes of K. lactis, spontaneous orthovanadateresistant mutants were selected from two strains of opposite mating type (see Materials and methods). The mutants of both strains were
K. lactis mutants and glycosylation
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Table I. Yeast strains. Strain
Genotype
Source
MW278-20C CBS2359/152 CPV1 CPV2 CPV3 CPV4 CPV5 CPV6 CPV7 CPV8
MATα ade2-1 leu2 uraA1-1 lac4-8 MATα metA MATα ade2-1 leu2 uraA1-1 lac4-8 vga1 MATα ade2-1 leu2 uraA1-1 lac4-8 vga2 MATα ade2-1 leu2 uraA1-1 lac4-8 vga3 MATα ade2-1 leu2 uraA1-1 lac4-8 vga4 MATa metA vga1 MATa metA vga2 MATa metA vga3 MATa metA vga4
Wesolowsky-Louvel Wesolowsky-Louvel Spontaneous mutant from MW278-20C Spontaneous mutant from MW278-20C Spontaneous mutant from MW278-20C Spontaneous mutant from MW278-20C Spontaneous mutant from CBS2359/152 Spontaneous mutant from CBS2359/152 Spontaneous mutant from CBS2359/152 Spontaneous mutant from CBS2359/152
isolated on YPD plates containing 7 mM of sodium orthovanadate (NaV); the minimal inhibitory concentration of NaV for the wild type strains was 5 mM. The vanadate-resistant colonies from MW278-20C (MATα) and CBS 2359/152 (MATa) appeared at a frequency of approximately 10–7. Among them, ten mutants of each strain were randomly chosen to determine the recessiveness or dominance of the mutations. To this aim, we crossed the mutants derived from MW278-20C with the CBS 2359/152 wild type strain and vice versa for the mutants derived from CBS 2359/152. The diploids were then analysed for their capability to grow on rich medium containing 7 mM NaV; all the diploid strains were unable to grow after 4 days of incubation, indicating that the vanadate-resistance mutations tested were recessive. In order to verify whether the mutants isolated had a single mutation, the same diploids were sporulated on malt extract and the resulting asci dissected. Ten asci were typically analysed for each diploid. The segregation of phenotypes VanR:VanS was always 2:2; this is the expected behaviour of single mutations (data not shown). The mutants were mated together in a combinatory procedure to determine the number of complementation groups represented. The diploids were then assayed for vanadate resistance and glycosylation defects of invertase. The results of the complementation analysis are presented in figure 1. The mutations analysed were assigned to four complementation groups and
each group contained more than one isolate. The four groups were named vga1 to vga4 (for vanadate glycosylation affected, see next section) and a representative isogenic strain from each group (CPV1 to CPV4; see table I) was used therein for the characterisation of the mutations. We first analysed the level of vanadate resistance. As shown in table II, CPV1 was the most resistant, CPV2 and CPV3 grew well up to 8 mM NaV. Since the vanadate-resistant mutants of S. cerevisiae have often been reported to be hypersensitive to aminoglycosides [8], we
Figure 1. Native gel electrophoresis of the active invertase from vga mutants and complementation between vga mutants based on invertase mobility. Lane 1, MW278-20C, wild type; lane 2, CPV1 × CPV6 diploid; lane 3, CPV1 × CPV7 diploid; lane 4, CPV1 × CPV8 diploid; lane 5, CPV2 × CPV7 diploid; lane 6, CPV2 × CPV8 diploid; lane 7, CPV3 × CPV8 diploid; lane 8, CPV4 haploid; lane 9, CPV3 haploid; lane 10, CPV2 haploid; lane 11, CPV1 haploid; lane 12, CBS 2359/152, wild type.
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Table II. Phenotypic analysis of vanadate-resistant mutants. Strains MW278-20C CPV1 CPV2 CPV3 CPV4
NaV (mM)
HygB (µg/mL)
Thermosensitivity
Calcofluor (µg/mL)
7
10
1.5
2.5
5
36 °C
37 °C
38 °C
10
15
40
– +++ +++ +++ +++
– ++ +/– +/– +
+++ +/– ++ ++ +++
+++ +/– +/– + +++
+++ +/– – – +++
+++ +/– + – +++
+++ – – – +
+ – – – –
+++ +++ +++ – +++
+++ – – – +++
+/– – – – +++
Growth of the cells was scored as very strong (+++), strong (++), weak (+), papillae (+/–) or none (–). The thermosensitivity test was performed comparing the growth of the cell at the indicated temperatures with respect to growth at 24 °C.
tested the resistance to hygromycin B (HygB) of vga mutants. The wild type K. lactis strains we studied were not able to grow at HygB concentrations above 40 µg/mL (not shown). The results reported in table II show that the vanadate-resistant mutants of K. lactis were affected to a variable extent in the resistance to HygB. An exception was CPV4; the resistance to HygB of this mutant was indistinguishable from that of the parental strain; in fact, CPV4 was able to grow in the presence of 40 µg/mL of HygB. We then studied the temperature dependence of the growth of vga mutants in YPD medium. The wild type strain was still able to grow, albeit slowly, at 38 °C, whereas the mutants were inhibited. Moreover, the CPV1, CPV2 and CPV3 strains failed to grow at 36 °C, whereas the CPV4 mutant was able to grow at this temperature and did not grow at 38 °C (see table II). Mutations altering N-linked glycosylation (see next section) are expected to affect the cell wall mannoproteins travelling trough the secretory apparatus. This may result in alterations of the cell wall structure that can be revealed by changes in the resistance to calcofluor white. We analysed the growth of mutants in YPD supplemented with various concentrations of this dye. As shown in table II, we observed sensitivity to calcofluor for all but one vga mutant, with CPV3 being more sensitive with respect to the others. The CPV4 mutant was resistant up to 40 µg/mL of calcofluor, while the wild type grew well up to 30 µg/mL.
Taken all together, the results in table II show that the four complementation groups had distinctive phenotypic traits. 3.2. Analysis of glycosylation defects in vga mutants
We made use of invertase as a reporter protein in order to investigate the defects in the N-linked glycosylation pathway of our mutants since, in S. cerevisiae, this protein can only be N-glycosylated [25]. The selected strains CPV1 to CPV4, representative of each complementation group, were analysed for the production of active invertase by native gel electrophoresis. Wild type cells secreted a population of glycosylated invertase visible as a diffuse band (figure 1, lanes 1 and 12); this was due to the heterogeneous size of the mannose outer chains N-linked to the secreted invertase [3]. As shown in figure 1, lanes 9, 10 and 11, strains with the vga1, vga2 and vga3 mutations produced only a fast-migrating form of invertase, representing the core- or ER-glycosylated form of this enzyme [12]. The CPV4 strain secreted slightly underglycosylated forms of invertase relative to wild type cells (figure 1, lane 8). We also analysed the invertase secreted from diploids obtained by crossing the mutants in a combinatory scheme. The results reported in figure 1, lanes 2 to 7, show that each combination of the studied mutations resulted in the production of fully glycosylated invertase forms having the same electrophoretic mobility as those produced by the wild type strains.
K. lactis mutants and glycosylation
We observed at least two different defects in the electrophoretic profile of the secreted invertase; this indicated that different steps of the N-linked glycosylation pathway were impaired in the analysed mutants. We then analysed the O-linked glycosylation of our mutants, employing as a reporter the chitinase. This protein is secreted from yeast cells into the growth medium and is exclusively O-mannosylated [16, 20]. Chitinase can be purified from the culture supernatant by selective chitin binding. Subsequent analysis by SDSPAGE is based on the assumption that the electrophoretic mobility of chitinase depends on the amount of the O-linked mannose [16]. Results reported in figure 2 show that the chitinase secreted by the CPV4 cells had greater mobility than that of the wild type counterpart (compare lanes 2 and 3), suggesting that the amount of O-linked mannose is reduced in this strain. The CPV3 mutant instead produced a chitinase with mobility indistinguishable from that of the wild type parent (compare lanes 1 and 3). This indicates that O-linked glycosylation of the CPV3 cells is probably unaffected; on the other hand, this mutant is strongly impaired in the N-linked glycosylation pathway (see figure 1). The chitinase was not detected in the culture supernatant of CPV1 and CPV2 strains; this could be ascribed to defects in the secretory pathway and remains to be investigated. 3.3. The K. lactis vga mutants are impaired in the polarized deposition of chitin
Alterations in the glycosylation pathway are expected to affect the production and delivery of enzymes and glycoproteins devoted to the organisation of the cell wall. The chitin, a linear β-1,4-N-acetylglucosamine polymer, is a minor but highly structured component of the yeast cell wall; it is mostly found in the bud scar, the ring-like structure that marks the bud emergence site. The localized deposition of chitin results from a highly organized delivery of various proteins through the polarised secretion apparatus.
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Figure 2. Immunoblot analysis of chitinase secreted from isogenic vga mutants. Lane 1, CPV3; lane 2, CPV4; lane 3, MW278-20C, wild type. Molecular mass marker on the right, plain arrow.
We thus investigated the morphology of chitin organisation in our mutants using fluorescence microscopy of calcofluor white stained cells. The results are reported in figure 3. The wild type cells showed the typical localisation of chitin at the neck between mother cells and emerging buds and at the rings of the bud scars (figure 3, panel A). The cultures of CPV1, CPV2 and CPV3 mutants (figure 3, panels B, C and D, respectively) exhibited a clumpy aspect and remarkable heterogeneity in cell morphology. A vast majority of the cells were large and round; the so-called ’Mickey Mouse’ shape was also present. The larger cells had a bright fluorescence staining pattern uniformly distributed all around the cell wall; in the smaller cells, chitin accumulation at the actively growing sites was still visible, together with calcofluor stained material diffused all around the cell wall. All these observations indicate that the localized deposition of chitin is impaired in these mutants; this defect is often referred to as loss of polarized growth. The CPV4 mutant (figure 3, panel E) exhibited different morphological changes, with the chitin staining pattern and cell shape very similar to the wild type ones; the cell size of the mutant was, however, larger than normal. The changes in the cell shape and size we observed in all the mutants studied were not due to osmotic defects, since they were not affected by the addition of 1 M sorbitol to the medium (results not shown).
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Figure 3. Analysis of chitin localisation in the isogenic vga mutants by fluorescence microscopy. Cells were grown to late exponential phase in YPD and stained with calcofluor white. All the pictures were at the same magnification. A. MW278-20C, wild type. B. CPV1. C. CPV2. D. CPV3. E. CPV4.
4. Discussion The mechanism of resistance to sodium orthovanadate in yeast cells is not completely understood; however, S. cerevisiae-resistant mutants isolated by four laboratories [4, 14, 22, 32] demonstrate a strong correlation of the vanadate-resistant phenotype with alterations in the glycosylation processes. We thus isolated spontaneous orthovanadate-resistant mutants of K. lactis affected in the glycosylation pathway of this biotechnologically significant yeast. These mutants fall into at least four complementation groups, and all of them exhibit defects in the N-linked glycosylation pathway, as revealed
by the electrophoretic profiles of the secreted invertase. This approach, proposed by Ballou [3], can reveal alterations in the mannose outer chain elongation of the secreted proteins, and this modification is a landmark event of the Golgi apparatus [4]. One of our mutants is also impaired in O-linked glycosylation, as indicated by the electrophoretic mobility shift of chitinase [16]. The K. lactis mutants we isolated are therefore affected in various glycosylation steps that presumably occur in the early phases of the secretory process. Dean [8] reported that S. cerevisiae mutants defective in all aspects of glycosylation are
K. lactis mutants and glycosylation
specifically sensitive to aminoglycosides, and suggested that the degree of sensitivity is related to the severity of glycosylation abnormalities. Our analysis reveals that K. lactis mutants present different degrees of sensitivity to HygB, with the exception of the CPV4 mutant that has the same HygB resistance level as the wild type parent. On the other hand, the CPV4 strain has an intermediate glycosylation profile, similar to the vrg4 and alg4 mutants of S. cerevisiae [19, 22], but both vrg4 and alg4 mutants are hypersensitive to HygB. This trait of the CPV4 strain could possibly offer novel insights into the still unclear link between glycosylation defects and sensitivity to aminoglycosides. Changes in the glycosylation processes could affect the secretion and delivery machinery impinging upon the organisation of the outer cell structures. The involvement of the genes identified by the analysed mutations in the assembly of the cell wall is supported by the altered resistance to calcofluor white and by fluorescence microscopy observations. We found both calcofluor hypersensitive (CPV1, CPV2 and CPV3) as well as resistant phenotypes (CPV4). Hypersensitivity to calcofluor is associated with delocalisation of chitin all along the cell wall. The deposition of chitin at the sites of active growth results from the accurate localisation of chitin synthase activities [7] also in coordination with several other proteins such as the septins [9]. Mondesert and co-workers [19] recently reported that N-linked glycosylation processes, together with polarization of the secretory machinery, control polarized growth and cell wall morphogenesis in S. cerevisiae. Mutants with altered glycosylation described here suggest that a similar connection can be established in K. lactis, and confirm that the mechanism of resistance to orthovanadate involves the glycosylation pathway. The characteristics of the CPV4 mutant are somewhat unusual: this mutant has a chitin localisation indistinguishable from that of the wild type by fluorescence microscopy and is hyperresistant to calcofluor white. The rare calcofluor-resistant mutants of S. cerevisiae are usually characterised by a decrease in chitin in
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the cell wall and in the activity of chitin synthase III, as reported for cal2 [27] or dit101 mutants, where the chitin is almost undetectable [21]. Resistance to the toxic compound thus results from the reduction or from the absence of the target molecule on the cell surface. In the CPV4 mutant of K. lactis, a novel resistance mechanism could be present; we are tempted to speculate that the glycosylation changes occurring in this mutant could affect glycoprotein(s) present in the cell wall architecture that interact with chitin. The described mutants should represent basic tools for the molecular genetics and cell biology studies of the glycosylation processes in K. lactis. Résumé — Mutants de Kluyveromyces lactis, altération de la glycosylation protéinique et morphogenèse pariétale. Chez la levure intéressante en biotechnologie, Kluyveromyces lactis, nous avons isolé des mutants spontanés, résistants à l’orthovanate sodique. La résistance est du type récessif chez tous les mutants étudiés. Quatre gènes ont été définis par complémentation, de vga1 à vga4. Ces mutants sont défectifs pour la glycosylation liée à N aussi bien qu’à O. De plus, ils sont sensibles à l’hygromicine B, un aminoglycoside, et au calcofluor, à l’exception du mutant vga4 qui se développe en présence de l’antibiotique mais qui résiste au calcofluor. Les mutations entraînent des altérations de la structure pariétale, révélée par une délocalisation de la chitine, des modifications de la forme et de la taille cellulaires et un aspect compact des cultures. Les mutants isolés peuvent servir d’outils de base dans l’analyse moléculaire et cellulaire des processus de glycosylation chez K. lactis. © Elsevier, Paris glycosylation / morphogenèse de la paroi cellulaire / Kluyveromyces lactis / mutant / chitine / vanadate
Acknowledgments We are indebted to Dr. Wesolowsky-Louvel for K. lactis wild type strains and helpful advice, and to Dr. Tanner for antibodies. We also thank Mr. Castelli for excellent technical assistance. This work was supported by the CNR Target Project on Biotechnology no. 97.01162.49, by the Commission of the European Community
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(BIO4-CT96-0003) and by MURST-University La Sapienza Cofin 1997.
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