CHARACTERIZATION OF MEMBRANE-BOUND SMALL GTP-BINDING PROTEINS FROM NICOTIANA-TABACUM

We have cloned nine cDNAs encoding small GTP-binding proteins from lea f cDNA libraries of tobacco (Nicotiana tabacum). These cDNAs encode di stinct proteins (22-25 kD) that display different levels of identity w ith members of the mammalian Rab family: Nt-Rab6 with Rab6 (83%), Nt-R ab7a-c with Rab7 (63-70%), and Nt-Rab11a-e with Rab11 (53-69%). Functi onally important regions of these proteins, including the ''effector b inding'' domain, the C-terminal Cys residues for membrane attachment, and the four regions involved in CTP-binding and hydrolysis, are highl y conserved. Northern and western blot analyses show that these genes are expressed, although at slightly different levels, in all plant tis sues examined. We demonstrate that the plant Rab5, Rab6, and Rab11 pro teins, similar to their mammalian and yeast counterparts, are tightly bound to membranes and that they exhibit different solubilization char acteristics. furthermore, we show that the yeast GTPase-activating pro tein Gyp6, shown to be specifically required to control the CTP hydrol ysis of the yeast Ypt6 protein, could interact with tobacco GTP-bindin g proteins. It increases in vitro the CTP hydrolysis rate of the wild- type Nt-Rab7 protein. In addition, it also increases, at different lev els, the CTP hydrolysis rates of a Nt-Rab7m protein with a Rab6 effect or domain and of two other chimaeric Nt-Rab6/Nt-Rab7 proteins. However , it does not interact with the wild-type Nt-Rab6 protein, which is mo st similar to the yeast Ypt6 protein.