Overmeyer J H, Wilson A L, Maltese W A
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614-5804, USA.
J Biol Chem. 2001 Jun 8;276(23):20379-86. doi: 10.1074/jbc.M101511200. Epub 2001 Mar 16.
The targeting of various Rab proteins to different subcellular compartments appears to be determined by variable amino acid sequences located upstream from geranylgeranylated cysteine residues in the C-terminal tail. All nascent Rab proteins are prenylated by geranylgeranyltransferase II, which recognizes the Rab substrate only when it is bound to Rab escort protein (REP). After prenylation, REP remains associated with the modified Rab until it is delivered to the appropriate subcellular membrane. It remains unclear whether docking of the Rab with the correct membrane is solely a function of features contained within the prenylated Rab itself (with REP serving as a "passive" carrier) or whether REP actively participates in the targeting process. To address this issue, we took advantage of a mutation in the alpha2 helix of Rab1B (i.e. Y78D) that abolishes REP and GDI interaction without disrupting nucleotide binding or hydrolysis. These studies demonstrate that replacing the C-terminal GGCC residues of Rab1B(Y78D) with a CLLL motif permits this protein to be prenylated by geranylgeranyltransferase I but not II both in cell-free enzyme assays and in transfected cells. Subcellular fractionation and immunofluorescence studies reveal that the prenylated Rab1B(Y78D)CLLL, which remains deficient in REP and GDI association is, nonetheless, delivered to the Golgi and endoplasmic reticulum (ER) membranes. When the dominant-negative S22N mutation was inserted into Rab1B-CLLL, the resulting monoprenylated construct suppressed ER --> Golgi protein transport. However, when the Y78D mutation was added to the latter construct, its inhibitory effect on protein trafficking was lost despite the fact that it was localized to the ER/Golgi membrane. Therefore, protein interactions mediated by the alpha2 helical domain of Rab1B(S22N) appear to be essential for its functional interaction with components of the ER --> Golgi transport machinery.
各种Rab蛋白靶向不同亚细胞区室的过程，似乎是由位于C末端尾巴中香叶基香叶基化半胱氨酸残基上游的可变氨基酸序列所决定。所有新生的Rab蛋白都由香叶基香叶基转移酶II进行异戊二烯化修饰，该酶仅在Rab蛋白与Rab护送蛋白（REP）结合时才识别Rab底物。异戊二烯化修饰后，REP仍与修饰后的Rab蛋白结合，直至其被转运至合适的亚细胞膜。目前尚不清楚Rab蛋白与正确膜的对接是否仅仅是异戊二烯化修饰后的Rab蛋白自身所含特征的功能（REP作为“被动”载体），还是REP积极参与了靶向过程。为了解决这个问题，我们利用了Rab1B的α2螺旋中的一个突变（即Y78D），该突变消除了REP和GDP解离抑制因子（GDI）的相互作用，而不破坏核苷酸结合或水解。这些研究表明，在无细胞酶分析和转染细胞中，用CLLL基序取代Rab1B（Y78D）的C末端GGCC残基，可使该蛋白被香叶基香叶基转移酶I而非II进行异戊二烯化修饰。亚细胞分级分离和免疫荧光研究表明，异戊二烯化修饰后的Rab1B（Y78D）CLLL虽然仍缺乏与REP和GDI的结合，但仍被转运至高尔基体和内质网（ER）膜。当将显性负性S22N突变插入Rab1B - CLLL时，所得的单异戊二烯化构建体抑制了内质网向高尔基体的蛋白质转运。然而，当将Y78D突变添加到后一种构建体中时，尽管它定位于内质网/高尔基体膜，但其对蛋白质运输的抑制作用却丧失了。因此，Rab1B（S22N）的α2螺旋结构域介导的蛋白质相互作用，似乎对其与内质网向高尔基体运输机制成分的功能相互作用至关重要。
