Leung G K, Schmidt W K, Bergo M O, Gavino B, Wong D H, Tam A, Ashby M N, Michaelis S, Young S G
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California 94141-9100, USA.
J Biol Chem. 2001 Aug 3;276(31):29051-8. doi: 10.1074/jbc.M102908200. Epub 2001 Jun 8.
Genetic studies in Saccharomyces cerevisiae identified two genes, STE24 and RCE1, involved in cleaving the three carboxyl-terminal amino acids from isoprenylated proteins that terminate with a CAAX sequence motif. Ste24p cleaves the carboxyl-terminal "-AAX" from the yeast mating pheromone a-factor, whereas Rce1p cleaves the -AAX from both a-factor and Ras2p. Ste24p also cleaves the amino terminus of a-factor. The mouse genome contains orthologues for both yeast RCE1 and STE24. We previously demonstrated, with a gene-knockout experiment, that mouse Rce1 is essential for development and that Rce1 is entirely responsible for the carboxyl-terminal proteolytic processing of the mouse Ras proteins. In this study, we cloned mouse Zmpste24, the orthologue for yeast STE24 and showed that it could promote a-factor production when expressed in yeast. Then, to assess the importance of Zmpste24 in development, we generated Zmpste24-deficient mice. Unlike the Rce1 knockout mice, Zmpste24-deficient mice survived development and were fertile. Since no natural substrates for mammalian Zmpste24 have been identified, yeast a-factor was used as a surrogate substrate to investigate the biochemical activities in membranes from the cells and tissues of Zmpste24-deficient mice. We demonstrate that Zmpste24-deficient mouse membranes, like Ste24p-deficient yeast membranes, have diminished CAAX proteolytic activity and lack the ability to cleave the amino terminus of the a-factor precursor. Thus, both enzymatic activities of yeast Ste24p are conserved in mouse Zmpste24, but these enzymatic activities are not essential for mouse development or for fertility.
在酿酒酵母中进行的遗传学研究鉴定出了两个基因，STE24和RCE1，它们参与从以CAAX序列基序结尾的异戊二烯化蛋白上切割三个羧基末端氨基酸。Ste24p从酵母交配信息素a因子上切割羧基末端的“-AAX”，而Rce1p则从a因子和Ras2p上切割-AAX。Ste24p还切割a因子的氨基末端。小鼠基因组包含酵母RCE1和STE24的直系同源基因。我们之前通过基因敲除实验证明，小鼠Rce1对发育至关重要，并且Rce1完全负责小鼠Ras蛋白的羧基末端蛋白水解加工。在本研究中，我们克隆了小鼠Zmpste24，它是酵母STE24的直系同源基因，并表明它在酵母中表达时可以促进a因子的产生。然后，为了评估Zmpste24在发育中的重要性，我们培育了Zmpste24基因缺陷小鼠。与Rce1基因敲除小鼠不同，Zmpste24基因缺陷小鼠在发育过程中存活下来并且具有生育能力。由于尚未鉴定出哺乳动物Zmpste24的天然底物，因此使用酵母a因子作为替代底物来研究Zmpste24基因缺陷小鼠的细胞和组织膜中的生化活性。我们证明，Zmpste24基因缺陷小鼠的膜与Ste24p基因缺陷酵母的膜一样，CAAX蛋白水解活性降低，并且缺乏切割a因子前体氨基末端的能力。因此，酵母Ste24p的两种酶活性在小鼠Zmpste24中得以保留，但这些酶活性对小鼠发育或生育能力并非必不可少。
