Plasmalemmal vesicles (PVs) or caveolae are plasma membrane invaginations and associated vesicles of regular size and shape found in most mammalian cell types. They are particularly numerous in the continuous endothelium of certain microvascular beds (e.g., heart, lung, and muscles) in which they have been identified as transcytotic vesicular carriers. Their chemistry and function have been extensively studied in the last years by various means, including several attempts to isolate them by cell fractionation from different cell types. The methods so far used rely on nonspecific physical parameters of the caveolae and their membrane (e.g., size-specific gravity and solubility in detergents) which do not rule out contamination from other membrane sources, especially the plasmalemma proper. We report here a different method for the isolation of PVs from plasmalemmal fragments obtained by a silica-coating procedure from the rat lung vasculature. The method includes sonication and flotation of a mixed vesicle fraction, as the first step, followed by specific immunoisolation of PVs on anticaveolin-coated magnetic microspheres, as the second step. The mixed vesicle fraction is thereby resolved into a bound subfraction (B), which consists primarily of PVs or caveolae, and a nonbound subfraction (NB) enriched in vesicles derived from the plasmalemma proper. The results so far obtained indicate that some specific endothelial membrane proteins (e.g., thrombomodulin, functional thrombin receptor) are distributed about evenly between the B and NB subfractions, whereas others are restricted to the NB subfraction (e.g., angiotensin converting enzyme, podocalyxin). Glycoproteins distribute unevenly between the two subfractions and antigens involved in signal transduction [e.g., annexin II, protein kinase Ca, the Ga subunits of heterotrimeric G proteins (as, aq, ai2, ai3)

Abstract. From rat livers labeled in vivo for 30 min with [35S] cys-met, we have isolated two classes of vesicular carders operating between the Golgi complex and the basolateral (sinusoidal) plasmalemma. The starting preparation is a Golgi light fraction (GLF) isolated by flotation in a discontinuous sucrose density gradient and processed through immunoisolation on magnetic beads coated with an antibody against the last 11 aa. of the pIgA-R tail. GLF and the ensuing subfractions (bound vs nonbound) were lysed, and the lysates processed through immunoprecipitation with anti-pIgA-R and anti-albumin antibodies followed by radioactivity counting, SDS-PAGE, and fluorography. The recovery of newly synthesized pIgA-R was >90% and the distribution was 90% vs 10% in the bound vs nonbound subfractions, respectively. Albumin radioactivity was recovered to ,x,80%, with 20% and 80% in bound vs nonbound subfractions, respectively.Other proteins studied were: (a) secretoryapolipoprotein-B, prothrombin,'C3 component of the complement, and caeruloplasmin; (b) membranetransferrin receptor, EGR-receptor, asialoglycoprotein receptor, and the glucose transporter. In all the experiments we have performed, the secretory proteins distributed up to 85 % in the nonbound subfraction (large secretory vacuoles), whereas the membrane proteins were segregated up to 95 % in the bound subfraction (small vesicular carders). These results suggest that in hepatocytes, membrane and secretory proteins are transported from the Golgi to the basolateral plasmamalemma by separate vesicular carders as in glandular cells capable of constitutive and regulated secretion.

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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, ‫؍‬ 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, ‫؍‬ 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 [␣-32 P]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.