Abstract
Mg2+ inhibits GDP release from Rab5WT but not from Rab5S34N, a mutant lacking Ser34 critical for Mg2+ coordination in the nucleotide binding pocket. Thus, inhibition of GDP release is apparently exerted via coordination of Mg2+ between Rab5 and GDP. Mg2+ also induces conformational changes in Rab5WT, demonstrated by increased tryptophan fluorescence intensity and a red shift in lambda max for the GDP-bound protein. Mg(2+)-induced fluorescence changes are not observed for Rab5S34N. The correlation between Mg2+ effects on nucleotide exchange and the fluorescence properties of Rab5 suggests that a conformation promoted through Mg2+ coordination with Ser34 also contributes to inhibition of GDP release. The role of structural changes in GDP release was investigated using C- and N-terminal truncation mutants. Similar to Rab5WT, Mg2+ inhibits GDP release and alters the fluorescence of Rab5(1-198) but only partially inhibits release from Rab5(23-198) and fails to induce changes in the latter's fluorescence properties. Since Rab5(23-198) maintains Ser34 necessary for Mg2+ coordination, the lack of Mg(2+)-induced fluorescence changes suggests a requirement for the N-terminal domain to promote a conformation blocking GDP release. A model for mechanisms of interaction between Ras-like proteins and their exchange factors is proposed.
Highlights
Rab proteins are a family of Ras-like small molecular weight GTPases that are localized to distinct subcellular compartments [1, 2] and believed to regulate specific steps of intracellular membrane trafficking [3,4,5,6]
The functional cycle of Rab proteins involves the delivery of the GDP-bound forms to the target membrane by a GDP dissociation inhibitor (GDI)1 [7,8,9], the exchange of GDP for GTP at membrane surface catalyzed by a guanine nucleotide exchange factor (GEF) [8, 9] and the retrieval of the GDP-bound forms from the membrane by GDI after GTP hydrolysis and membrane fusion [7]
Based on the correlation between inhibition of GDP release and conformational changes promoted by Mg2ϩ, we propose that in vivo, Mg2ϩ prevents GDP dissociation from Rab5 until a guanine nucleotide exchange factor acts to promote GDP/GTP exchange, perhaps through interactions with the GTP-binding protein’s N-terminal domain
Summary
Rab proteins are a family of Ras-like small molecular weight GTPases that are localized to distinct subcellular compartments [1, 2] and believed to regulate specific steps of intracellular membrane trafficking [3,4,5,6]. Important functional roles of the N-terminal domains of several Ras-like GTP-binding proteins have been noted in studies of guanine nucleotide exchange [33,34,35,36,37,38]. Deletion of the N-terminal domain of Rab results in a loss of function (34 –37) and interferes with the protein’s post-translational processing [37] These observations suggest that N-terminal domains of Ras-like GTP-binding proteins may participate in the regulation of guanine nucleotide exchange and represent crucial structural domains necessary for the function of the proteins. Based on the correlation between inhibition of GDP release and conformational changes promoted by Mg2ϩ, we propose that in vivo, Mg2ϩ prevents GDP dissociation from Rab until a guanine nucleotide exchange factor acts to promote GDP/GTP exchange, perhaps through interactions with the GTP-binding protein’s N-terminal domain
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