Effect of a pmr1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba α-galactosidase by Saccharomyces cerevisiae

Harmsen, Michiel M.; Langedijk, Annette C; Tuinen, E van; Geerse, Ruud H.; Raué, Hendrik A.; Maat, Jaap

doi:10.1016/0378-1119(93)90318-w

Cited by 47 publications

(22 citation statements)

References 29 publications

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“…Genes encoding P-type Ca'+-ATPases of the early secretory pathway have been isolated from a variety of organisms; the Ca2+ pump encoded by the P M R l gene of S. cerevisiae is perhaps the best characterized (Rudolph et a[., 1989;Harmsen et al, 1993;Sorin et a/., 1997;Diirr et al, 1998). This protein localizes in the membranes of the Golgi apparatus (Antebi & Fink, 1992) and inactivation of P M R l gave rise to a complex set of phenotypes.…”

Section: U C C E L L E T T I a N D O T H E R Smentioning

confidence: 99%

“…This protein localizes in the membranes of the Golgi apparatus (Antebi & Fink, 1992) and inactivation of P M R l gave rise to a complex set of phenotypes. Mutant cells exhibited calcium-related growth defects (Rudolph et al, 1989), reduced glycosylation of secreted invertase (Rudolph et al, 1989;Diirr et al, 1998), enhanced release of heterologous proteins (Moir et al 1987;Harmsen et al, 1993Harmsen et al, , 1997 and defective sorting of carboxypeptidase Y (Antebi & Fink, 1992). Recent studies have demonstrated that Mn2+ can partially substitute for the Ca2+ requirement and that PmrIp can operate as a common transporter for Ca'+ and Mn2+ (Diirr et al, 1998).…”

Section: U C C E L L E T T I a N D O T H E R Smentioning

confidence: 99%

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Inactivation of the KlPMR1 gene of Kluyveromyces lactis results in defective cell-wall morphogenesis

et al. 1999

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The P-type Ca2+-ATPases are the transporters responsible for calcium homeostasis in the cell compartments of eukaryotes. The KlPMR7 gene of KIuyveromyces lactis encodes a P-type Ca*+-ATPase, which is functionally and structurally homologous to Pmrl p of Saccharomyces cerevisiae, the calcium pump localized in the Golgi membranes. In this work, a novel involvement of KlPmrlp in cell-wall morphogenesis of K, lactis is reported. KlpmrIA cells exhibited the loss of outer-chain extension in the glycosylation of secreted proteins. The absence of KlPmrlp resulted in the accumulation of round, large cells with an abnormally thick cell wall, as revealed by transmission electron microscopy. The deletant strain also showed a delocalized deposition of chitin in the lateral cell wall accompanied by an unbalanced ratio of insoluble to soluble glucans. These morphological defects were accompanied by the presence of irregularly shaped nuclei and by a DNA content greater than 2n. Addition of I 0 mM Ca2+ to the medium of the KlpmrIA strain reversed the chitin-deposition impairment, recovered the alteration to the glucan ratio and restored a normal thickness of the cell wall. The mutant cells resumed wildtype size, shape and nuclear morphology but the DNA content indicated the persistence of defects in the co-ordination between DNA replication and cell division. The glycosylation defects were completely unaffected by the calcium supplement. These results indicate that calcium homeostasis controlled by KlPmrlp plays an important role in the cell-wall morphogenesis of K. lactis.

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Section: U C C E L L E T T I a N D O T H E R Smentioning

confidence: 99%

Section: U C C E L L E T T I a N D O T H E R Smentioning

confidence: 99%

Inactivation of the KlPMR1 gene of Kluyveromyces lactis results in defective cell-wall morphogenesis

et al. 1999

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“…Alterations in cell calcium homeostasis have been observed in an S. cerevisiae PMR1-disrupted mutant; PMR1 encodes a Ca 2ϩ -ATPase that is localized in the Golgi apparatus (3,15). The inactivation of this gene also resulted in a significant increase in the secretion of several heterologous proteins (26,37). We could thus speculate that the oversecretion phenotype of the dgr151-1 mutant could also be linked to alterations in cell Ca 2ϩ homeostasis that, in turn, originate from a perturbed balance of Glc-1-P and Glc-6-P.…”

Section: Discussionmentioning

confidence: 99%

Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

Donnini

Farina

Neglia

et al. 2004

Appl Environ Microbiol

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The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1␤ compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position ؉527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N-and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.Yeasts are very useful hosts for the production of heterologous proteins. The yeast Kluyveromyces lactis presents several advantages over other yeast species. It is positive for lactose fermentation, is able to grow on cheap substrates such as residual whey from dairy industries, and has competitive secretory properties, excellent large-scale fermentation characteristics, and food grade status; also, both episomal and integrative expression vectors are available for it (for reviews, see references 20, 40, and 50). Its ability to secrete heterologous proteins into the medium at a concentration higher than that secreted by Saccharomyces cerevisiae was demonstrated previously (50), although the secretory and glycosylation processes and their regulation are still poorly understood for K. lactis (1,42,43).For K. lactis, the regulation of primary carbon metabolism differs markedly from that for S. cerevisiae and reflects the dominance of respiration over fermentation that is typical for the majority of yeast species (7). In K. lactis, respiration is not repressed by glucose, and fermentative and oxidative metabolism can take place simultaneously. Glucose repression, however, does exist: several enzymes that are required for alternate carbohydrate metabolism have been shown to be subject to glucose repression (6,13,17,25,30). The K. lactis genes involved in glucose repression include RAG1, encoding a lowaffinity glucose permease (23, 48); DGR151 (or RAG5), encoding the single hexokinase of this yea...

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“…pUR4536 is derived from pHEN (16) and contains the lacI q gene and unique restriction sites to allow cloning of the llama V HH genes. pUR4548 is derived from pSY1 (14). The BstEII site in the leu2 gene was removed from this plasmid by PCR, and the cloning sites between the SUC2 signal sequence and the terminator were replaced in order to facilitate the cloning of the V HH gene fragments.…”

Section: Methodsmentioning

confidence: 99%

Bactericidal Effects of a Fusion Protein of Llama Heavy-Chain Antibodies Coupled to Glucose Oxidase on Oral Bacteria

Szynol

Soet

Tuyl

et al. 2004

Antimicrob Agents Chemother

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Enzymes such as lactoperoxidase and glucose oxidase (GOx) are used as antimicrobial agents in oral care products. Their low specificities and substantiveness can be reduced by covalent coupling of antimicrobial molecules to antibodies. Variable domains (V HH ) derived from llama heavy-chain antibodies are particularly suited for such an approach. The antibodies are composed solely of heavy-chain dimers; therefore, production of active fusion proteins by using molecular biology-based techniques is less complicated than production by use of conventional antibodies. In this study, a fusion protein consisting of V HH and GOx was constructed and expressed by Saccharomyces cerevisiae. A llama was immunized with Streptococcus mutans strain HG982. Subsequently, B lymphocytes were isolated and cDNA fragments encoding the V HH fragments were obtained by reverse transcription-PCR. After construction of a V HH library in Escherichia coli and screening of the library against mutans group streptococci and Streptococcus sanguinis strains, we found two V HH fragments with high specificities for S. mutans strains. A GOx gene was linked to the two V HH genes and cloned into S. cerevisiae yeasts. The yeasts expressed and secreted the recombinant proteins into the growth medium. The test of binding of fusion proteins to oral bacteria through their V HH fragments showed that S. mutans had been specifically targeted by GOx-S120, one of the fusion protein constructs. A low concentration of the fusion protein was also able to selectively kill S. mutans within 20 min in the presence of lactoperoxidase and potassium iodide. These findings demonstrate that the fusion protein GOx-V HH is potentially valuable in the selective killing of target bacteria such as S. mutans.

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Effect of a pmr1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba α-galactosidase by Saccharomyces cerevisiae

Cited by 47 publications

References 29 publications

Inactivation of the KlPMR1 gene of Kluyveromyces lactis results in defective cell-wall morphogenesis

Inactivation of the KlPMR1 gene of Kluyveromyces lactis results in defective cell-wall morphogenesis

Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

Bactericidal Effects of a Fusion Protein of Llama Heavy-Chain Antibodies Coupled to Glucose Oxidase on Oral Bacteria

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