Molecular cloning and subcellular localization of three GTP-binding proteins of the rab subfamily

Small GTPases of the rab subfamily are involved in regulation of intracellular membrane transport events. We recently used a PCR approach to isolate short cDNA fragments of a number of novel rab sequences. These PCR fragments have not been used with cDNA library screening and PCR-based techniques to clone the cDNAs encoding three of these proteins, rab12, rab22, and rab24. By northern blot analysis, the messages were found to be present in a wide variety of mouse tissues. However, quantitative differences in the mRNA levels between the tissues were detected. We determined the subcellular localization of the GTPases by expressing the c-myc epitope-tagged proteins with the Semliki Forest virus and the vaccinia T7 vector systems. Transiently expressed rab12 was localized to the Golgi complex. This localization was confirmed using a polyclonal anti-peptide antibody detecting the endogenous protein in BHK cells. rab22 expressed from the cDNA was localized to endosomal compartments and to the plasma membrane. After longer periods of expression, the protein was found on abnormally large perinuclear endosomal structures, suggesting that it is a potent regulator of events in the endocytic pathway. Finally, rab24 was found in the endoplasmic reticulum/cis-Golgi region and on late endosomal structures. The localization of rab24 may indicate its involvement in autophagy-related processes.