Rab1a and multiple other Rab proteins are associated with the transcytotic pathway in rat liver.
To better understand the function of Rab1a, we have immunoisolated Rab1a-associated transport vesicles from rat liver using affinity-purified anti-Rab1a-coated magnetic beads. A fraction enriched in endoplasmic reticulum (ER) to Golgi transport vesicles (CV2, rho = 1.158) was subjected to immunoisolation, and proteins of the bound and non-bound subfractions were analyzed by Western blotting. To our surprise, we found that immunoisolated vesicles contained not only ER markers (105-kDa form of the polymeric IgA receptor (pIgAR)) but also transcytotic markers (dIgA and the 120-kDa form of pIgAR), suggesting that Rab1a is associated with transcytotic vesicles in rat liver. To investigate this possibility, we used an antibody to the cytoplasmic domain of pIgAR to immunoisolate transcytotic vesicles from a fraction (CV1, rho = 1. 146) known to be enriched in these vesicles. Rab1a was detected in the immunoadsorbed subfractions. The composition of the vesicles immunoisolated from the CV1 fraction on anti-Rab1a was similar to that of transcytotic vesicles immunoisolated from the same fraction on pIgAR. Both were enriched in transcytotic markers and depleted in ER and Golgi markers. The main difference between the two was that those isolated on anti-Rab1a appeared to be enriched in postendosomal transcytotic vesicles, whereas those isolated on pIgAR contained both pre- and postendosomal elements. Analysis of anti-Rab1a isolated vesicles using [alpha-32P]GTP overlay demonstrated the presence of multiple GTP-binding proteins. Some of these were identified by immunoblotting as epithelia-specific Rab17 and ubiquitous Rabs1b, -2, and -6. Taken together, these results indicate that: 1) Rab1a is associated with both ER to Golgi and postendosomal transcytotic vesicles, and 2) multiple GTP-binding proteins are associated with each class of isolated vesicle.
Jin
M
M
Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093, USA.
Saucan
L
L
Farquhar
M G
MG
Palade
G E
GE
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000378:metabolism; D020761:rab1 GTP-Binding Proteins
D002352:Carrier Proteins; D019204:GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins