Shapiro A D, Pfeffer S R
Department of Biochemistry, Stanford University School of Medicine, California 94305-5307, USA.
J Biol Chem. 1995 May 12;270(19):11085-90. doi: 10.1074/jbc.270.19.11085.
Rab9 is a Ras-like GTPase required for the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Rab9 occurs in the cytosol as a complex with GDP dissociation inhibitor (GDI), which we have shown delivers prenyl Rab9 to late endosomes in a functional form. We report here basal rate constants for guanine nucleotide dissociation and GTP hydrolysis for prenyl Rab9. Both rate constants were influenced in part by the hydrophobic environment of the prenyl group. Guanine nucleotide dissociation and GTP hydrolysis rates were lower in the presence of lipid; detergent stimulated intrinsic nucleotide exchange. GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 2.4-fold. GDI-alpha associated with prenyl Rab9 with a KD of 60 nM in 0.1% Lubrol and 23 nM in 0.02% Lubrol. In 0.1% Lubrol, GDI-alpha inhibited GDP dissociation half maximally at 72 +/- 18 nM, consistent with the KD determinations. These data suggest that GDI-alpha associates with prenyl Rab9 with a KD of < or = 23 nM under physiological conditions. Finally, a previously uncharacterized minor form of GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 1.9-fold and bound prenyl Rab9 with a KD of 67 nM in 0.1% Lubrol.
Rab9是一种类Ras GTP酶，在晚期内体和反式高尔基体网络之间运输甘露糖6 - 磷酸受体时发挥作用。Rab9以与GDP解离抑制剂（GDI）形成的复合物形式存在于细胞质中，我们已经证明GDI能将异戊二烯化的Rab9以功能形式递送至晚期内体。我们在此报告异戊二烯化Rab9的鸟嘌呤核苷酸解离和GTP水解的基础速率常数。这两个速率常数部分受异戊二烯基团疏水环境的影响。在脂质存在的情况下，鸟嘌呤核苷酸解离和GTP水解速率较低；去污剂刺激内在核苷酸交换。GDI-α抑制异戊二烯化Rab9的GDP解离达2.4倍。在0.1% Lubrol中，GDI-α与异戊二烯化Rab9结合的解离常数（KD）为60 nM，在0.02% Lubrol中为23 nM。在0.1% Lubrol中，GDI-α在72±18 nM时半最大程度抑制GDP解离，与KD测定结果一致。这些数据表明，在生理条件下，GDI-α与异戊二烯化Rab9结合的KD≤23 nM。最后，一种先前未被表征的GDI-α的次要形式抑制异戊二烯化Rab9的GDP解离达1.9倍，在0.1% Lubrol中与异戊二烯化Rab9结合的KD为67 nM。
