Membrane targeting of Rab GTPases is influenced by the prenylation motif
Membrane targeting of Rab GTPases is influenced by the prenylation motif
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DOI：
10.1091/mbc.e02-10-0639
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发表时间：
2003-05-01
影响因子：
3.3
通讯作者：
Seabra, MC
中科院分区：
文献类型：
--
作者：
Gomes, AQ;Ali, BR;Seabra, MC
Rab GTPases 是膜运输的调节剂。 Rab 在涉及 Rab 护卫蛋白和 Rab 香叶基香叶基转移酶的反应中通过（通常）连接两个香叶基香叶基基团与靶膜特异性结合。相反，相关的 GTP 酶被 CAAX 异戊二烯基转移酶单独异戊二烯化。我们报道，二香叶基香叶基修饰对于 Rab5a 和 Rab27a 分别靶向内体和黑素体非常重要。含有两个异戊二烯化半胱氨酸（CGC、CC、CCQNI 和 CCA）的 EGFP-Rab5 突变体在 HeLa 细胞中的瞬时表达不影响包膜体靶向或功能，而单半胱氨酸突变体（CSLG、CVLL 或 CVIM）被错误定位到内质网。 （ER）并且无法正常工作。类似地，Rab27aCVLL 突变体也错误定位到 ER，并且 Rab27a 无效背景 (Rab27a(ash)) 上的转基因表达没有挽救毛色表型，表明 Rab27aCVLL 在体内不起作用。 CAAX 异戊二烯基转移酶抑制和温度变化实验进一步表明，单次或双重修饰的 Rab 通过 Rab 护送蛋白/Rab 香叶基香叶基转移酶依赖性机制被招募到膜上，该机制不同于包含 CAAX 的 GTPases 的插入。最后，我们表明 RabGDIalpha 从膜中提取单修饰和双修饰的 Rab，并提出 Rab 错误定位到 ER 是由于定位信息丢失造成的。
Rab GTPases are regulators of membrane traffic. Rabs specifically associate with target membranes via the attachment of (usually) two geranylgeranyl groups in a reaction involving Rab escort protein and Rab geranylgeranyl transferase. In contrast, related GTPases are singly prenylated by CAAX prenyl transferases. We report that di-geranylgeranyl modification is important for targeting of Rab5a and Rab27a to endosomes and melanosomes, respectively. Transient expression of EGFP-Rab5 mutants containing two prenylatable cysteines (CGC, CC, CCQNI, and CCA) in HeLa cells did not affect enclosomal targeting or function, whereas mono-cysteine mutants (CSLG, CVLL, or CVIM) were mistargeted to the endoplasmic reticulum. (ER) and were nonfunctional. Similarly, Rab27aCVLL mutant is also mistargeted to the ER and transgenic expression on a Rab27a null background (Rab27a(ash)) did not rescue the coat color phenotype, suggesting that Rab27aCVLL is not functional in vivo. CAAX prenyl transferase inhibition and temperature-shift experiments further suggest that Rabs, singly or doubly modified are recruited to membranes via a Rab escort protein/Rab geranylgeranyl transferase-dependent mechanism that is distinct from the insertion of CAAX-containing GTPases. Finally, we show that both singly and doubly modified Rabs are extracted from membranes by RabGDIalpha and propose that the mistargeting of Rabs to the ER results from loss of targeting information.