Trueblood C E, Boyartchuk V L, Picologlou E A, Rozema D, Poulter C D, Rine J
Molecular and Cell Biology Department, University of California, Berkeley, California 94720, USA.
Mol Cell Biol. 2000 Jun;20(12):4381-92. doi: 10.1128/MCB.20.12.4381-4392.2000.
Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a(1), the a(2), or the X position of the a-factor Ca(1)a(2)X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a(1) position, V, L, I, C, or M at the a(2) position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a(1) substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.
许多含有羧基末端CaaX序列基序的蛋白质，包括Ras和酵母a因子，会经历一系列连续的翻译后加工步骤。在半胱氨酸最初进行异戊二烯化之后，C末端的三个氨基酸会被蛋白水解去除，新形成的异戊二烯化半胱氨酸会被羧甲基化。构成CaaX序列的特定氨基酸会影响蛋白质是否能够进行异戊二烯化和蛋白水解。在本研究中，我们评估了在a因子Ca(1)a(2)X序列CVIA的a(1)、a(2)或X位置具有所有可能单氨基酸取代的a因子变体的加工情况。在体内研究了两种已知的酵母CaaX蛋白酶Afc1p和Rce1p的底物特异性。Afc1p和Rce1p都能够对在a(1)位置为A、V、L、I、C或M、在a(2)位置为V、L、I、C或M或者在X位置为半胱氨酸异戊二烯化可接受的任何氨基酸的a因子进行蛋白水解。另外八个具有a(1)取代的a因子变体可被Rce1p蛋白水解，但不能被Afc1p蛋白水解。相反，Afc1p能够对Rce1p可能无法蛋白水解的其他a因子变体进行蛋白水解。体外试验表明，对于在野生型AFC1 RCE1菌株中未产生可检测晕圈的11个a因子变体，法尼基化受损或无法检测到。在RCE1中分离出改善a因子-CAMQ蛋白水解的突变，表明Rce1p中的氨基酸取代E139K、F189L和Q201R影响了其底物特异性。
