Immunoisolation and partial characterization of endothelial plasmalemmal vesicles (caveolae).

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 C alpha, the G alpha subunits of heterotrimeric G proteins (alpha s, alpha q, alpha i2, alpha i3), small GTP-binding proteins, endothelial nitric oxide synthase, and nonreceptor protein kinase c-src] are concentrated in the NB (plasmalemma proper-enriched) subfraction rather than in the caveolae of the B subfraction. Additional work should show whether discrepancies between our findings and those already recorded in the literature represent inadequate fractionation techniques or are accounted for by chemical differentiation of caveolae from one cell type to another.

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.

Uptake and incorporation of an epitope-tagged sialic acid donor into intact rat liver Golgi compartments. Functional localization of sialyltransferase overlaps with beta-galactosyltransferase but not with sialic acid O-acetyltransferase.

The transfer of sialic acids (Sia) from CMP-sialic acid (CMP-Sia) to N-linked sugar chains is thought to occur as a final step in their biosynthesis in the trans portion of the Golgi apparatus. In some cell types such Sia residues can have O-acetyl groups added to them. We demonstrate here that rat hepatocytes express 9-O-acetylated Sias mainly at the plasma membranes of both apical (bile canalicular) and basolateral (sinusoidal) domains. Golgi fractions also contain 9-O-acetylated Sias on similar N-linked glycoproteins, indicating that O-acetylation may take place in the Golgi. We show here that CMP-Sia-FITC (with a fluorescein group attached to the Sia) is taken up by isolated intact Golgi compartments. In these preparations, Sia-FITC is transferred to endogenous glycoprotein acceptors and can be immunochemically detected in situ. Addition of unlabeled UDP-Gal enhances Sia-FITC incorporation, indicating a substantial overlap of beta-galactosyltransferase and sialyltransferase machineries. Moreover, the same glycoproteins that incorporate Sia-FITC also accept [3H]galactose from the donor UDP-[3H]Gal. In contrast, we demonstrate with three different approaches (double-labeling, immunoelectron microscopy, and addition of a diffusible exogenous acceptor) that sialyltransferase and O-acetyltransferase machineries are much more separated from one another. Thus, 9-O-acetylation occurs after the last point of Sia addition in the trans-Golgi network. Indeed, we show that 9-O-acetylated sialoglycoproteins are preferentially segregated into a subset of vesicular carriers that concentrate membrane-bound, but not secretory, proteins.

Membrane and secretory proteins are transported from the Golgi complex to the sinusoidal plasmalemma of hepatocytes by distinct vesicular carriers.

From rat livers labeled in vivo for 30 min with [35S] cys-met, we have isolated two classes of vesicular carriers 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 approximately 80%, with 20% and 80% in bound vs nonbound subfractions, respectively. Other proteins studied were: (a) secretory-apolipoprotein-B, prothrombin, C3 component of the complement, and caeruloplasmin; (b) membrane-transferrin 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 carriers). These results suggest that in hepatocytes, membrane and secretory proteins are transported from the Golgi to the basolateral plasmalemma by separate vesicular carriers as in glandular cells capable of constitutive and regulated secretion.

Differential colchicine effects on the transport of membrane and secretory proteins in rat hepatocytes in vivo: bipolar secretion of albumin.

We carried out a comparative investigation on the effects of colchicine (25 mumoles/100 gm body wt) on the intracellular transport, processing and discharge by secretion or proteolytic processing of a membrane protein (i.e., the polymeric IgA receptor) and a secretory protein (i.e., albumin) in rat hepatocytes. The results obtained indicated the following: (a) the transport and processing of polymeric IgA receptor is strongly inhibited and delayed, but the appearance of secretory component in the bile is not arrested; (b) polymeric IgA receptor reaches the sinusoidal plasmalemma in colchicine-treated specimens, as it does in controls; (c) albumin discharge into the plasma is strongly inhibited and markedly delayed in colchicine-treated as compared with control animals; (d) the reverse applies for albumin secretion in the bile, which is increased by a large factor; (e) newly synthesized albumin secreted directly from hepatocytes in control and in colchicine-treated animals is the major source of bile albumin; and (f) colchicine affects in different ways the polymeric IgA receptor and albumin arrival at the sinusoidal front and especially at the biliary front of the hepatocyte.

Protein traffic between distinct plasma membrane domains: isolation and characterization of vesicular carriers involved in transcytosis.

We have isolated a population of vesicular carriers involved in the transport (transcytosis) of proteins from the basolateral to the apical plasma membrane of hepatocytes. The obtained fraction was enriched in compartments containing known transcytosed proteins and depleted in elements of the secretory pathway, Golgi elements, basolateral plasma membrane, as well as early endosomal components. The fraction was analyzed by biochemical and immunological procedures. Antibodies raised against the proteins in the fraction recognized a single 108K antigen. Based on its subcellular distribution, the 108K antigen may represent a novel marker for transcytotic vesicular carriers.