Weber Y, Santore U J, Ernst J F, Swoboda R K
Institut für Mikrobiologie, Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany.
J Bacteriol. 2001 Jan;183(1):46-54. doi: 10.1128/JB.183.1.46-54.2001.
Sec20p is a component of the yeast Saccharomyces cerevisiae secretory pathway that does not have a close homolog in higher eukaryotic cells. To verify the function of Sec20p in other fungal species, we characterized the gene encoding a Sec20p homolog in the human fungal pathogen Candida albicans. The deduced protein has 27% identity with, but is missing about 100 N-terminal residues compared to S. cerevisiae Sec20p, which is part of the cytoplasmic tail interacting with the cytoplasmic protein Tip20p. Because a strain lacking both C. albicans SEC20 alleles could not be constructed, we placed SEC20 under transcriptional control of two regulatable promoters, MET3p and PCK1p. Repression of SEC20 expression in these strains prevented (MET3p-SEC20 allele) or retarded (PCK1p-SEC20 allele) growth and led to the appearance of extensive intracellular membranes, which frequently formed stacks. Reduced SEC20 expression in the PCK1p-SEC20 strain did not affect morphogenesis but led to a series of hypersensitivity phenotypes including supersensitivity to aminoglycoside antibiotics, to nystatin, to sodium dodecyl sulfate, and to cell wall inhibitors. These results demonstrate the occurrence and function of Sec20p in a fungal species other than S. cerevisiae, but the lack of the N-terminal domain and the apparent absence of a close TIP20 homolog in the C. albicans genome also indicate a considerable diversity in mechanisms of retrograde vesicle traffic in eukaryotes.
Sec20p是酿酒酵母分泌途径的一个组成部分,在高等真核细胞中没有与之密切同源的蛋白。为了验证Sec20p在其他真菌物种中的功能,我们对人类真菌病原体白色念珠菌中编码Sec20p同源物的基因进行了表征。推导的蛋白质与酿酒酵母Sec20p有27%的同一性,但与酿酒酵母Sec20p相比,缺少约100个N端残基,Sec20p的这部分胞质尾与胞质蛋白Tip20p相互作用。由于无法构建缺失两个白色念珠菌SEC20等位基因的菌株,我们将SEC20置于两个可调控启动子MET3p和PCK1p的转录控制之下。在这些菌株中抑制SEC20表达会阻止(MET3p-SEC20等位基因)或延缓(PCK1p-SEC20等位基因)生长,并导致出现大量的细胞内膜,这些内膜经常形成堆叠。PCK1p-SEC20菌株中SEC20表达的降低不影响形态发生,但会导致一系列超敏表型,包括对氨基糖苷类抗生素、制霉菌素、十二烷基硫酸钠和细胞壁抑制剂的超敏反应。这些结果证明了Sec20p在酿酒酵母以外的真菌物种中的存在和功能,但白色念珠菌基因组中缺少N端结构域以及明显没有紧密的TIP20同源物,这也表明真核生物中逆行囊泡运输机制存在相当大的多样性。