RAB1A AND MULTIPLE OTHER RAB PROTEINS ARE ASSOCIATED TRANSCYTOTIC PATHWAY IN RAT-LIVER

To better understand the function of Rab1a, we have immunoisolated Rab 1a-associated transport vesicles from rat liver using affinity-purifie d anti-Rab1a-coated magnetic beads, A fraction enriched in endoplasmic reticulum (ER) to Golgi transport vesicles (CV2, rho = 1.158) was sub jected to immunoisolation, and proteins of the bound and non-bound sub fractions were analyzed by Western blotting, To our surprise, we found that immunoisolated vesicles contained not only ER markers (105-kDa f orm of the polymeric IgA receptor (pIgAR)) but also transcytotic marke rs (dIgA and the 120-kDa form of pIgAR), suggesting that Rab1a is asso ciated with transcytotic vesicles in rat liver, To investigate this po ssibility, we used an antibody to the cytoplasmic domain of pIgAR to i mmunoisolate transcytotic vesicles from a fraction (CV1, rho = 1.146) known to be enriched in these vesicles, Rab1a was detected in the immu noadsorbed subfractions. The composition of the vesicles immunoisolate d from the CV1 fraction on anti-Rab1a was similar to that of transcyto tic 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 post-endosomal transcytotic vesicles, whereas those isolated on pIgAR contained both pre- and postendosomal elements, Analysis of anti-Rab1a isolated vesicles using [alpha-P-32] GTP overlay demonstrated the presence of multiple GTP-binding proteins , Some of these were identified by immunoblotting as epithelia-specifi c Rab17 and ubiquitous Rabs1b, -2, and -6, Taken together, these resul ts indicate that: 1) Rab1a is associated with both ER to Gels and post endosomal transcytotic vesicles, and 2) multiple GTP-binding proteins are associated with each class of isolated vesicle.