RESUMO
The plant enzyme xyloglucan endotransglycosylase (XET; EC 2.4.1.207, xyloglucan:xyloglucosyl transferase) participates in selective modification of plant cell walls during cell growth. XETs are potential catalysts in various applications. Here, sequences encoding two XETs from Gerbera hybrida and Betula pendula are reported. The encoded proteins, which are 51% identical at the amino acid level, were expressed in the yeast Pichia pastoris in secreted form with the aid of mating factor alpha signal sequence. XET production in shake flask cultivations was better at 22 degrees C than at 30 degrees C. Both the yield of protein of expected molecular mass and the XET activity improved at the lower temperature. Under all cultivation conditions studied, higher amounts of XET from B. pendula (BXET) were expressed than XET from G. hybrida (GXET). Both XET enzymes were produced in 16l fed-batch bioreactor cultures. GXET was produced in methanol-limited fed-batch cultivation in minimal medium, and BXET in temperature-limited fed-batch (TLFB) in minimal or complex medium. Production was highest in TLFB in complex medium. BXET was purified from the culture filtrate and characterized. Based on the specific activity of the purified protein, 60-70 mg l(-1) BXET was produced in the TLFB in complex medium.
Assuntos
Asteraceae/enzimologia , Betula/enzimologia , Biotecnologia/métodos , Glicosiltransferases/biossíntese , Pichia/enzimologia , Sequência de Aminoácidos , Asteraceae/genética , Betula/genética , Biomassa , Reatores Biológicos , Técnicas de Cultura de Células , Meios de Cultura , Filtração , Expressão Gênica , Glicosiltransferases/análise , Glicosiltransferases/genética , Glicosiltransferases/isolamento & purificação , Glicosiltransferases/metabolismo , Metanol/metabolismo , Dados de Sequência Molecular , Pichia/genética , Pichia/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
The SEB1/SBH1 and the SSO genes encode components of the protein secretory machinery functioning at the opposite ends, ER translocation and exocytosis, respectively, of the secretory pathway in Saccharomyces cerevisiae. Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired in protein secretion. Furthermore, their overexpression in wild-type yeast enhances production of secreted proteins in S. cerevisiae, which suggests that they may be rate-limiting factors in this process. Here we report isolation of Kluyveromyces lactis homologues of these genes. KlSSO1 and KlSEB1 were isolated as clones capable of rescuing growth of ts sso2-1 and seb1Delta seb2Delta sem1Delta strains, respectively, at the restrictive temperature. The encoded Kluyveromyces proteins are up to 70% identical with the S. cerevisiae homologues at the amino acid level and can functionally replace them. Interestingly, KlSSO1 and KlSEB1 show similar enhancing effect on production of a secreted protein as the SSO and SEB1 genes of S. cerevisiae when overexpressed. In accordance with the high homology level of the secretory pathway proteins in different yeast species, the polyclonal antibodies raised against S. cerevisiae Seb1p, Sso2p and Sec4p can detect homologous proteins in cell lysates of K. lactis and Pichia pastoris, the latter also in Candida utilis. The GenBank Accession Nos are AF307983 (K. lactis SSO1) and AF318314 (K. lactis SEB1).
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Kluyveromyces/genética , Proteínas de Membrana/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Western Blotting , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Exocitose/fisiologia , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Kluyveromyces/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Plasmídeos , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , alfa-Amilases/metabolismoRESUMO
The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane. Previously, we identified SEB1/SBH1, encoding the beta subunit of the Sec61p ER translocation complex, as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex. Here we show the functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery. We show that overexpression of SEB1 suppresses the growth defect in all exocyst sec mutants. In addition, overexpression of SEC61 or SSS1 encoding the other two components of the Sec61p complex suppressed the growth defects of several exocyst mutants. Seb1p was coimmunoprecipitated from yeast cell lysates with Sec15p and Sec8p, components of the exocyst complex, and with Sec4p, a secretory vesicle associated Rab GTPase that binds to Sec15p and is essential for exocytosis. The interaction between Seb1p and Sec15p was abolished in sec15-1 mutant and was restored upon SEB1 overexpression. Furthermore, in wild type cells overexpression of SEB1 as well as SEC4 resulted in increased production of secreted proteins. These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst.
