Farnsworth C C, Seabra M C, Ericsson L H, Gelb M H, Glomset J A
Howard Hughes Medical Institute, University of Washington, Seattle 98195.
Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11963-7. doi: 10.1073/pnas.91.25.11963.
Rab proteins are Ras-related small GTPases that are geranylgeranylated on cysteine residues located at or near their C termini. They differ from other geranylgeranylated small GTPases in several important respects. (i) Most Rab proteins contain two adjacent cysteine residues within one of the following C-terminal sequence motifs: -XXCC, -XCXC, or -CCXX; (ii) a Rab protein that ends in a -XCXC motif has been shown to be geranylgeranylated on both adjacent cysteine residues; and (iii) Rab proteins are substrates of a unique Rab-specific geranylgeranyltransferase. Whether this enzyme catalyzes the geranylgeranylation of both cysteines is unknown. We addressed this question by direct structural analysis of in vitro prenylated proteins. We incubated recombinant Rab geranylgeranyltransferase, Rab escort protein, and [1-3H]geranylgeranyl pyrophosphate with recombinant wild-type Rab1A (-XXCC), Rab3A (-XCXC), or Rab5A (-CCXX) and treated each labeled protein with trypsin. We then analyzed the resulting peptides by HPLC and electrospray mass spectrometry and found that for each protein both C-terminal adjacent cysteines were geranylgeranylated. These results indicate that Rab geranylgeranyltransferase/Rab escort protein catalyzes the geranylgeranylation of both cysteines in Rab proteins with three distinct C-terminal motifs and suggest that other Rab proteins with these motifs may be similarly modified.
Rab蛋白是与Ras相关的小GTP酶，在其C末端或附近的半胱氨酸残基上发生香叶基香叶基化。它们在几个重要方面不同于其他香叶基香叶基化的小GTP酶。（i）大多数Rab蛋白在以下C末端序列基序之一中包含两个相邻的半胱氨酸残基：-XXCC、-XCXC或-CCXX；（ii）已证明以-XCXC基序结尾的Rab蛋白在两个相邻的半胱氨酸残基上都发生了香叶基香叶基化；（iii）Rab蛋白是一种独特的Rab特异性香叶基香叶基转移酶的底物。这种酶是否催化两个半胱氨酸的香叶基香叶基化尚不清楚。我们通过对体外异戊烯化蛋白进行直接结构分析来解决这个问题。我们将重组Rab香叶基香叶基转移酶、Rab护送蛋白和[1-³H]香叶基香叶基焦磷酸与重组野生型Rab1A（-XXCC）、Rab3A（-XCXC）或Rab5A（-CCXX）一起孵育，并用胰蛋白酶处理每种标记蛋白。然后我们通过HPLC和电喷雾质谱分析所得肽段，发现对于每种蛋白，两个C末端相邻的半胱氨酸都发生了香叶基香叶基化。这些结果表明，Rab香叶基香叶基转移酶/Rab护送蛋白催化具有三种不同C末端基序的Rab蛋白中两个半胱氨酸的香叶基香叶基化，并表明具有这些基序的其他Rab蛋白可能会被类似地修饰。
