LOCALIZATION OF THE SMALL GTP-BINDING PROTEIN RAB1P TO EARLY COMPARTMENTS OF THE SECRETORY PATHWAY

We have studied the localization of the small GTPase rab1p in differen t cell types using polyclonal antibodies prepared against the rab1A is oform of the protein. Immunofluorescence microscopy of normal rat kidn ey (NRK) and mouse myeloma cells showed the association of the protein with the Golgi complex and peripheral sites where it colocalized with p58, a pre- and cis-Golgi marker protein. Rab1p and p58 also had simi lar distributions in membrane fractions derived from rat pancreas micr osomes. Both were concentrated in two intermediate density subfraction s between the rough endoplasmic reticulum and trans-Golgi, whereas rab 6p, previously localized to middle and trans-Golgi, was enriched in th e light density trans-Golgi fraction, Immunoperoxidase electron micros copy of NRK and myeloma cells revealed the association of rab1p with 1 -2 cisternae, vacuolar, and tubulovesicular membranes in the cis-Golgi region, The rab1p-specific staining typically covered the entire late ral surface of the cisternae but, in weakly stained cells, local label ing between closely opposed membranes could also be seen, The rab1p-po sitive preGolgi compartment had a predominantly tubulovesicular appear ance in NRK cells whereas in myeloma cells it consisted of vacuoles su rrounded by rab1p-positive vesicles and tubules of heterogenous size, In both cell types the rough ER cisternae and the nuclear envelope con tained negligible labeling and no continuities between these and the r ab1p-positive membranes were observed. In addition, in myeloma cells t he smooth ER subcompartment, containing endogenous retrovirus particle s, was devoid of rab1p-labeling. These results indicate that the pre-G olgi (intermediate) compartment consists of different membrane domains and its morphology can vary considerably between different cell types , Further, they suggest that the recruitment of rab1p to membranes occ urs predominantly in a post-ER location and that the protein functions in targeting/fusion events within the pre- and cis-Golgi membranes.