FUNCTIONAL AND STRUCTURAL INTERACTIONS OF THE RAB5 D136N MUTANT WITH XANTHINE NUCLEOTIDES

Rab5 is a Ras-related GTPase which regulates endosomal fusion. The D13 6N mutant of Rab5, which was predicted to switch specificity from guan ine to xanthine nucleotides, was expressed in E. coli, extracted with urea, purified by column chromatography, and refolded by stepwise dial ysis against buffer containing XDP. The purified protein bound xanthin e nucleotides with considerably higher affinity than guanine nucleotid es. In vitro prenylation of the mutant protein was highly dependent on xanthosine diphosphate. In contrast, both the wild type and mutant pr oteins were protected from proteolysis equally well by non-cognate and cognate triphosphate nucleosides at high concentration. The D136N Rab 5 mutant appears to be a valuable reagent in conjunction with xanthine nucleotides for the study of protein-nucleotide interactions in syste ms in which multiple GTPases are active, although interactions with no n-cognate nucleotides should be evaluated if they are present at high concentration. (C) 1995 Academic Press. Inc.