Abstract
A 3.85-kb genomic fragment containing the KlPCL1 gene, with an open reading frame (ORF) of 1359 bp, was isolated from Kluyveromyces lactis genomic library by heterologous colony hybridization using the Saccharomyces cerevisiae PRC1 (ScPRC1) gene as a probe. The KlPCL1 nucleotide sequence was identical to the KLLAOC17490g ORF of K. lactis and showed >55 % identity with S. cerevisiae YBR139w and PRC1 genes encoding carboxypeptidases. The deduced KlPcl1p amino acid sequence displayed strong similarities to yeast and higher eukaryotic carboxypeptidases. In silico analyses revealed that KlPcl1p contained several highly conserved regions characteristic of the serine-type carboxypeptidases, such as the catalytic triad in the active site and the LNGGPGCSS, FHIAGESYAGHYIP and ICNWLGN motifs involved in the substrate binding. All this suggests that the KlPCL1 gene product belongs to the serine carboxypeptidase family. Sporulation and ascus dissection of a diploid strain heterozygous for single-copy disruption of KlPCL1 revealed that this gene is not essential in K. lactis. Further analyses of haploid and diploid deletion mutants demonstrated that disruption of the KlPCL1 gene neither impaired sporulation nor affected growth abilities of K. lactis cells under a variety of physiological conditions, e.g., growth on different carbon sources, at various temperatures or pH of the medium, and under nitrogen depletion.
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Abbreviations
- CPY:
-
carboxypeptidase Y
- PCR:
-
polymerase chain reaction
- G418:
-
geneticin
- PRC:
-
proteinase C
- Kl :
-
Kluyveromyces lactis
- SM:
-
synthetic medium
- ME:
-
malt extract (medium)
- Sc :
-
Saccharomces cerevisiae
- MM:
-
minimal medium
- YP:
-
yeast extract-peptone
- ORF:
-
open reading frame
- YPD:
-
yeast extract-peptone-dextrose
- PCL:
-
proteinase C-like
References
Baxter S.M., Rosenblum J.S., Knutson S., Nelson M.R., Montimurro J.S., Di Gennaro J.A., Speir J.A., Burbaum J.J., Fet-Row J.S.: Synergistic computational and experimental proteomics approaches for more accurate detection of active serine hydrolases in yeast. Mol.Cell.Proteomics 3, 209–225 (2004).
Bendtsen J.D., Nielsen H., von Heijne G., Brunak S.: Improved prediction of signal peptides: signal P 3.0. J.Mol.Biol. 340, 783–795 (2004).
Bianchi M.M., Falcone C., Chen X.J., Weśolowski-Louvel M., Frontali L., Fukuhara H.: Transformation of the yeast Kluyveromyces lactis by new vectors derived from the 1.6 μm circular plasmid pKD1. Curr.Genet. 12, 185–192 (1987).
Billard P., Ménart S., Blaisonneau J., Bolotin-Fukuhara M., Fukuhara H., Weśolowski-Louvel M.: Glucose uptake in Kluyveromyces lactis. Role of the HGT1 gene in glucose transport. J.Bacteriol. 178, 5860–5866 (1996).
Brown A.J., Planta R.J., Restuhadi F., Bailey D., Butler P., Cadahia J., Cerdan M., De Jonge M., Gardner D., Gent M., Hayes A., Kolen C., Lombardia L., Murad A., Oliver R., Sefton M., Thevelein J., Tournu H., Van Delft Y., Verbart D., Winderickx J., Oliver S.: Transcript analysis of 1003 novel yeast genes using high-throughput Northern hybridizations. EMBO J. 20, 3177–3186 (2001).
Byrne K.P., Wolfe K.H.: Visualizing syntenic relationships among the hemiascomycetes with the Yeast Gene Order Browser. Nucl. Acids Res. 34 (Database issue), D452–D455 (2006).
Dujon B., Sherman D., Fisher G.: Genome evolution in yeasts. Nature 430, 35–44 (2004).
Flores M.V., Cuellas A., Voget C.E.: The proteolytic system of the yeast Kluyveromyces lactis. Yeast 15, 1437–1448 (1999).
Gasch A.P., Spellman P.T., Kao C.M., Carmel-Harel O., Eisen M.B., Storz G., Botstein D., Brown P.O.: Genomic expression programs in the response of yeast cells to environmental changes. Mol.Biol.Cell 11, 4241–4257 (2000).
Hayashi R., Moore S., Stein W.H.: Carboxypeptidase from yeast. Large scale preparation and the application to COOH-terminal analysis of peptides and proteins. J.Biol.Chem. 248, 2296–2302 (1973).
Van den Hazel H.B., Kielland-Brandt M.C., Winther J.R.: Review: biosynthesis and function of yeast vacuolar proteases. Yeast 12, 1–16 (1996).
Hoffman C.S., Winston F.: A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267–272 (1987).
Hussein L., Elasyed S., Foda S.: Reduction of lactose in milk by purified lactase produced by Kluyveromyces lactis. J.Food Prot. 52, 30–34 (1989).
Inoue H., Nojima H., Okayama H.M.: High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23–28 (1990).
Jones E.W., Webb G.C., Hiller M.A.: Biogenesis and function of yeast vacuole, pp. 363–470 in J.R. Pringle, J.R. Broach, E.W. Jones (Eds): The Molecular and Cellular Biology of the Yeast Saccharomyces, Vol. 3. Cold Spring Harbor, New York 1997.
Jung G., Ueno H., Hayashi R.: Carboxypeptidase Y: structural basis for protein sorting and catalytic triad. J.Biochem.(Tokyo) 126, 1–6 (1999).
Liao D.I., Remington S.J.: Structure of wheat serine carboxypeptidase II at 3.5-Å resolution. A new class of serine proteinase. J.Biol. Chem. 265, 6528–6531 (1990).
Lundblad V., Hartzog G., Moqtaderi Z.: Manipulation of cloned yeast DNA, unit 13.10 in F.M. Ausubel, R. Brent, R. Kingston, D. Moore, J. Seidman, J. Smith, K. Struhl (Eds): Current Protocols in Molecular Biology. John Wiley & Sons, New York 1989; Supplement 1997.
Nakase H., Murata S., Ueno H., Hayashi R.: Substrate recognition mechanism of carboxypeptidase Y. Biosci.Biotechnol.Biochem. 65, 2465–2471 (2001).
Nasr F., Becam A.-M., Grzybowska E., Zagulski M., Slonimski P.P., Herbert C.J.: An analysis of the sequence of part of the right arm of chromosome II of S. cerevisiae reveals new genes encoding an amino-acid permease and a carboxypeptidase. Curr.Genet. 26, 1–7 (1994).
Van Ooyen A., Dekker P., Huang M., Olsthoorn M., Jacobs D., Colussi P., Taron C.: Heterologous protein production in the yeast Kluyveromyces lactis. FEMS Yeast Res. 6, 381–392 (2006).
Pearson B.M., Hernando Y., Schweizer M.: Construction of PCR-ligated long flanking homology cassettes for use in the functional analysis of six unknown open reading frames from the left and right arms of Saccharomyces cerevisiae chromosome XV. Yeast 14, 391–399 (1998).
Sambrook J., Fritsch E.F., Maniatis T.: Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, New York 1989.
Stennicke H.R., Mortensen U.H., Breddam K.: Studies on the hydrolytic properties of (serine) carboxypeptidase Y. Biochemistry 35, 7131–7141 (1996).
Van den Berg J.A., Van der Laken K.J., Van Ooyen A., Renniers T., Rietveld K., Schaap A., Brake A., Bishop R., Schultz K., Moyer D., Richman M., Shuster J.R.: Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin. Bio/Technology 8, 135–139 (1990).
Wach A., Brachat A., Pohlmann R., Philippsen P.: New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808 (1994).
Weśolowski-Louvel M., Tanguy-Rougeau C., Fukuhara H.: A nuclear gene required for the expression of the linear DNA-associated killer system in the yeast Kluyveromyces lactis. Yeast 4, 71–81 (1988).
Weśolowski-Louvel M., Breunig K.D., Fukuhara H.: Kluyveromyces lactis, pp. 139–202 in K. Wolf (Ed.): Nonconventional Yeasts in Biotechnology: a Handbook. Springer-Verlag, Berlin 1996.
Winston F., Dollard C., Ricupero-Hovasse S.L.: Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11, 53–55 (1995).
Winzeler E., Shoemaker D., Astromoff A., Liang H., Anderson K., Andre B., Bangham R., Benito R., Boeke J., Bussey H., Chu A., Connelly C., Davis K., Dietrich F., Dow S., Bakkoury M., Foury F., Friend S., Gentalen E., Giaever G., Hegemann J., Jones T., Laub M., Liao H., Liebundguth N., Lockhart D., Lucau-Danila A., Lussier M., M’rabet N., Menard P., Mittmann M., Pai C., Rebischung C., Revuelta J., Riles L., Roberts C., Ross-Macdonald P., Scherens B., Snyder M., Sookhai-Mahadeo S., Storms R., Véronneau S., Voet M., Volckaert G., Ward T., Wy-Socki R., Yen G., Yu K., Zimmermann K., Philippsen P., Johnston M., Davis R.: Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).
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Staneva, D., Uccelletti, D., Venkov, P. et al. The Kluyveromyces lactis CPY homologous genes — Cloning and characterization of the KlPCL1 gene. Folia Microbiol 53, 325–332 (2008). https://doi.org/10.1007/s12223-008-0051-9
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DOI: https://doi.org/10.1007/s12223-008-0051-9