Sanford J, Codina J, Birnbaumer L
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1991 May 25;266(15):9570-9.
Lipid modifications that may be introduced into several subunits of G proteins were explored by in vitro translation of recombinant mRNAs in reticulocyte lysates. In agreement with studies by others, myristic acid was incorporated into alpha i's and alpha o, but not alpha s, beta, or gamma's. In contrast, mevalonate (Mev) was incorporated only into gamma-subunits. Both, the gamma-subunit of transducin (gamma T) and that of other G proteins (gamma G) were modified by the lysates but with different characteristics. Labeled gamma T was unstable and was rapidly proteolyzed. Labeled gamma G was stable. The Mev-derivative in gamma G was sensitive to methyliodide and, after cleavage and chromatographic analysis, comigrated with the C20 polyisoprenol geranylgeraniol. This indicated that gamma G had been geranylgeranylated and that this polyisoprenoid was attached to the protein through a thioether linkage. It is thought that polyisoprenylation is defined by the COOH-terminal sequence Cys-A-A-X, where A is an aliphatic acid and X is any amino acid. Replacement by mutation of the Cys of the COOH-terminal -Cys-Ala-Ile-Leu sequence of gamma G with Ser abolished Mev incorporation, suggesting this Cys as the site of attachment of the geranylgeranyl moiety. Yet, Mev incorporation was less than 10% as much into gamma G with the Cys-A-A-X sequence -Cys-Ala-Ile-Trp. Consistent with geranylgeranylation, the C15 farnesyl moiety of farnesyl pyrophosphate was not incorporated into gamma G unless the incubations were fortified with Mev. In contrast, the farnesyl moiety was incorporated in an Mev-independent manner into gamma T (COOH terminus: -Cys-Val-Ile-Ser) and c-Ha-ras (COOH terminus: -Cys-Val-Leu-Ser) which are both farnesylated rather than geranylgeranylated. Thus, 1) separate enzymes appear to be involved in transferring farnesyl and geranylgeranyl groups to proteins, 2) structural factors other than the CAAX box contribute to the activity of the polyisoprenylating enzymes, and 3) this type of lipidation may be part of a proteolytic signaling system. Polyisoprenylation, which increases hydrophobicity of the derivatized protein, may play a role in anchoring not only ras but also G proteins to membranes.
通过在网织红细胞裂解物中对重组mRNA进行体外翻译，研究了可能引入到G蛋白几个亚基中的脂质修饰。与其他人的研究一致，肉豆蔻酸被掺入αi和αo，但不掺入αs、β或γ亚基。相反，甲羟戊酸（Mev）仅掺入γ亚基。转导素的γ亚基（γT）和其他G蛋白的γ亚基（γG）都被裂解物修饰，但具有不同的特性。标记的γT不稳定，会迅速被蛋白酶水解。标记的γG稳定。γG中的Mev衍生物对甲基碘敏感，在裂解和色谱分析后，与C20聚异戊二烯香叶基香叶醇迁移率相同。这表明γG已被香叶基香叶基化，并且这种聚异戊二烯通过硫醚键与蛋白质相连。据认为，聚异戊二烯化由COOH末端序列Cys-A-A-X定义，其中A是脂肪酸，X是任何氨基酸。用Ser取代γG的COOH末端-Cys-Ala-Ile-Leu序列中的Cys，消除了Mev的掺入，表明该Cys是香叶基香叶基部分的附着位点。然而，对于具有Cys-A-A-X序列-Cys-Ala-Ile-Trp的γG，Mev的掺入量不到10%。与香叶基香叶基化一致，除非孵育中添加Mev，否则法尼基焦磷酸的C15法尼基部分不会掺入γG。相反，法尼基部分以不依赖Mev的方式掺入γT（COOH末端：-Cys-Val-Ile-Ser）和c-Ha-ras（COOH末端：-Cys-Val-Leu-Ser），它们都被法尼基化而不是香叶基香叶基化。因此，1）似乎有不同的酶参与将法尼基和香叶基香叶基基团转移到蛋白质上；2）除CAAX框外的结构因素有助于聚异戊二烯化酶的活性；3）这种类型的脂化可能是蛋白水解信号系统的一部分。聚异戊二烯化增加了衍生化蛋白质的疏水性，可能不仅在将ras而且在将G蛋白锚定到膜上起作用。
