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 different cell types using polyclonal antibodies prepared against the rab1A isoform of the protein. Immunofluorescence microscopy of normal rat kidney (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 similar distributions in membrane fractions derived from rat pancreas microsomes. Both were concentrated in two intermediate density subfractions between the rough endoplasmic reticulum and trans-Golgi, whereas rab6p, previously localized to middle and trans-Golgi, was enriched in the light density trans-Golgi fraction. Immunoperoxidase electron microscopy 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 lateral surface of the cisternae but, in weakly stained cells, local labeling between closely opposed membranes could also be seen. The rab1p-positive pre-Golgi compartment had a predominantly tubulovesicular appearance in NRK cells whereas in myeloma cells it consisted of vacuoles surrounded by rab1p-positive vesicles and tubules of heterogeneous size. In both cell types the rough ER cisternae and the nuclear envelope contained negligible labeling and no continuities between these and the rab1p-positive membranes were observed. In addition, in myeloma cells the smooth ER subcompartment, containing endogenous retrovirus particles, was devoid of rab1p-labeling. These results indicate that the pre-Golgi (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 occurs predominantly in a post-ER location and that the protein functions in targeting/fusion events within the pre- and cis-Golgi membranes.
Saraste
J
J
Department of Biochemistry and Molecular Biology, University of Bergen, Norway.
Lahtinen
U
U
Goud
B
B
D000818:Animals; D000906:Antibodies; D015415:Biomarkers / Q000032:analysis; D002458:Cell Fractionation; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D020558:GTP Phosphohydrolases / Q000032:analysis*; D019204:GTP-Binding Proteins / Q000032:analysis*; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure*; D007124:Immunoenzyme Techniques; D007668:Kidney; D051379:Mice; D016253:Microscopy, Immunoelectron; D008861:Microsomes / Q000378:metabolism / Q000648:ultrastructure*; D009101:Multiple Myeloma; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure; D010179:Pancreas / Q000378:metabolism / Q000648:ultrastructure*; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins; D020691:rab GTP-Binding Proteins
D000906:Antibodies; D015415:Biomarkers; D029701:Saccharomyces cerevisiae Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins