Abstract. An intrinsic membrane protein of brain synaptic vesicles with Mr 38,000 (p38, synaptophysin) has recently been partially characterized (Jahn, R., W. Schiebler, C. Ouimet, and P. Greengard, 1985, Proc. Natl. Acad. Sci. USA, 83:4137--4141;Wiedenmann, B., and W. W. Franke, 1985, Cell, 41:1017-1028. We have now studied the presence of p38 in a variety of tissues by light and electron microscopy immunocytochemistry and by immunochemistry. Our results indicate that, within the nervous system, p38, like the neuronspecific phosphoprotein synapsin I, is present in virtually all nerve terminals and is selectively associated with small synaptic vesicles (SSVs). No p38 was detectable on large dense-core vesicles (LDCVs). p38 and synapsin I were found to be present in similar concentrations throughout the brain. Outside the nervous system, p38 was found in a variety of neuroendocrine cells, but not in any other cell type. In neuroendocrine cells p38 was localized on a pleiomorphic population of small, smooth-surfaced vesicles, which were interspersed among secretory granules and concentrated in the Golgi area, but not on the secretory granules themselves. Immunoblot analysis of endocrine tissues and cell lines revealed a band with a mobility slightly different from that of neuronal p38. This difference was attributable to a difference in glycosylation.The finding that p38, like synapsin I, is a component of SSVs of virtually all neurons, but not of LDCVs, supports the idea that SSVs and LDCVs are organelles of two distinct pathways for regulated neuronal secretion. In addition, our results indicate the presence in a variety of neuroendocrine cells of an endomembrane system, which is related to SSVs of neurons but is distinct from secretory granules.

Abstract. The endocytosis of SV-40 into CV-1 cells was studied using biochemical and ultrastructural techniques. The half-time of binding of [35S]methionineradiolabeled SV-40 to CV-1 cells was 25 min. Most of the incoming virus particles remained undegraded for several hours. Electron microscopy showed that some virus entered the endosomal/lysosomal pathway via coated vesicles, while the majority were endocytosed via small uncoated vesicles. After infection at high multiplicity, one third of total cell-associated virus was observed to enter the ER, starting 1-2 h after virus application. The viruses were present in large, tubular, smooth membrane networks generated as extentions of the ER. The results describe a novel and unique membrane transport pathway that allows endocytosed viral particles to be targeted from the plasma membrane to the ER.

Abstract. We have used a variety of immunocytochemical procedures to localize albumin in transit through the capillary endothdium of the murine myocardium and thereby identify endothelial cell structures involved in albumin efflux. The most informative results were obtained with a protocol that included (a) removal of endogenous albumin by perfusion of the heart with PBS supplemented with 14 mM glucose, (b) perfusion of the heart vasculature with exogenous (bovine) albumin for various short time periods, (c) fixation of the vessels by formaldehyde-glutaraldehyde mixtures, (d) processing of fixed myocardium specimens through L. R. White embedding followed by sectioning, or direct thin frozen sectioning, and (e) reacting the surface of such specimens with antialbumin antibodies followed by gold-labeled secondary antibodies. The results indicate that (a) monomeric albumin binds (with low affinity) to the luminal surface of the capillary endothelium, (b) it is restricted in transit through the endothelium to plasmalemmal vesicles, and (c) it appears in the pericapillary spaces <15 s after the beginning of its perfusion. No albumin concentration gradients, centered with their maxima on the exits from intercellular spaces, were detected at any time points, including the shortest ones (15 and 30 s) investigated. Additional information comparing monomeric vs. polymeric albumin transcytosis was obtained using albumin-gold complexes. The results are discussed in terms of vesicular transport of albumin across the endothelium and the relations of this type of transport to the postulated pore systems of the physiological literature.W P'ITHIN the cardiovascular system, albumin performs a variety of functions, including (a) maintenance of the oncotic pressure of the blood plasma (13), (b) regulation of the permeability of the microvasculature by forming a "fiber matrix" through interaction with the ectodomains of the proteins of the luminal plasmalemma of the endothelium and its associated structures (6), and (c) transport of fatty acids, steroids, hormones, and bilirubin within the circulation and between the plasma and interstitial fluid (2,5,27). In part, the carrier functions of albumin involve its own transport across the vascular endothelium, primarily at the level of the microvasculature as evidenced by its presence in varied but characteristic concentrations within the interstitial fluid and lymph associated with, or derived from, all microvascular beds.At present, the pathways, forces, and mechanisms involved in albumin transport from the blood plasma to the interstitial fluid are issues on which there is no general agreement. Data obtained in physiological experiments have been interpreted in terms of albumin moving by diffusion and convection through a system of small (~<12 nm diam) and large (20-50 nm diam) pores (10,15,20) widely assumed to be located in the intercellular junctions of the endothelium (13,20). Movement through these pores is presumably regulated by the fibrous matrix and controlled by concentration...

Introduction

The nature of second messenger-responsive intracellular Ca2+ stores in neurons remains open for discussion. Here, we demonstrate the existence in Purkinje cells (PCs) of endoplastic reticulum (ER) subcompartments characterized by an uneven distribution of three proteins involved in Ca2+ storage and release: the inositol 1,4,5-trisphosphate (InsP3) receptor, Ca(2+)-ATPase, and calsequestrin. Ca(2+)-ATPase and the InsP3 receptor have a widespread, although not identical, distribution throughout the ER. Calsequestrin is localized throughout the smooth ER and is particularly concentrated in pleiomorphic vesicles with a moderately electron-dense core, which appear to represent a subcompartment of the smooth ER. In double-labeling experiments many of these vesicles were unlabeled by InsP3 receptor antibodies. These results suggest a key role of the ER as an intracellular Ca2+ store and demonstrate a possible structural basis for distinct intracellular Ca2+ pools regulated by different second messengers.

Synaptophysin, an integral membrane protein of small synaptic vesicles, was expressed by transfection in fibroblastic CHO-Kl cells. The properties and localization of synaptophysin were compared between transfected CHO-Kl cells and native neuroendocrine PC12 cells. Both cell types similarly glycosylate synaptophysin and sort it into indistinguishable microvesicles. These become labeled by endocytic markers and are primarily concentrated below the plasmalemma and at the area of the Golgi complex and the centrosomes. A small pool of synaptophysin is transiently found on the plasma membrane. In CHO-Kl cells synaptophysin co-localizes with transferrin that has been internalized by receptor-mediated endocytosis. These findings suggest that synaptophysin in transfected CHO-Kl cells and neuroendocrine PC12 cells is directed into a pathway of recycling microvesicles which, in CHO cells, is shown to coincide with that of the transferrin receptor. They further indicate that fibroblasts have the ability to sort a synaptic vesicle membrane protein. Our results suggest a pathway for the evolution of small synaptic vesicles from a constitutively recycling organelle which is normally present in all cells.

Electron microscope autoradiography was used to study the cellular localization of seven glycoproteins rapidly cleared from the circulating plasma of rats and taken up by the liver. I and 15 min after intravenous administration of the 125I-glycoproteins, livers were fixed in situ by perfusion and processed for autoradiography. Autoradiographic grains in the developed sections were found to represent the intact 125 I-ligand. A quantitative analysis of the distribution and concentration (density) of autoradiographic grains over the three major cell types of the liver was then performed. Three molecules, asialo-fetuin, asialo-orosomucoid, and lactosaminated RNase A dimer, the oligosaccharide chains of which terminate in galactose residues, were bound and internalized almost exclusively (>900 /0) by hepatocytes. Conversely, four molecules, the oligosaccharide chains of which terminate in either N-acetyl-glucosamine (agalacto-orosomucoid) or mannose (ahexosamino-orosomucoid, preputial,8-glucuronidase, and mannobiosaminated RNase A dimer), were specifically bound and internalized by cells lining the blood sinusoids-that is, by Kupffer cells and endothelial cells. Endothelial cells were two to six times more active (on a cell volume basis) than were Kupffer cells in the internalization of these four 125 I-ligands. Mannose and N-acetylglucosamine-terminated glycoproteins competed with each other for uptake into either endothelial cells or Kupffer cells, indicating that a single system recognized mannose or Nacetyl-glucosamine residues. Finally, agalacto-orosomucoid and ahexosaminoorosomucoid were also associated with hepatocytes, but competition experiments utilizing excess asialo-orosomucoid demonstrated that residual galactosyl residues were responsible for this association.J. CELL BIOLOGY

To examine the physiological role of the GLUT4/muscle-fat specific facilitative glucose transporter in regulating glucose homeostasis, we have generated transgenic mice expressing high levels of this protein in an appropriate tissue-specific manner. Examination of two independent founder lines demonstrated that high-level expression of GLUT4 protein resulted in a marked reduction of fasting glucose levels (=70 mg/dl) compared to wild-type mice ("'130 mg/dl). Surprisingly, 30 min following an oral glucose challenge the GLUT4 transgenic mice had only a slight elevation in plasma glucose levels (Q"90 mg/dl), whereas wild-type mice displayed a typical 2-to 3-fold increase (==250-300 mg/dl). In parallel to the changes in plasma glucose, insulin levels were --2-fold lower in the transgenic mice compared to the wild-type mice. Furthermore, isolated adipocytes from the GLUT4 transgenic mice had increased basal glucose uptake and subcellular fractionation indicated elevated levels of cell surface-associated GLUT4 protein. Consistent with these results, in situ immunocytochemical localization of GLUT4 protein in adipocytes and cardiac myocytes indicated a marked increase in plasma membrane-associated GLUT4 protein in the basal state. Taken together these data demonstrate that increased expression of the human GLUT4 gene in vivo results in a constitutively high level of cell surface GLUT4 protein expression and more efficient metabolic control over fluctuations in plasma glucose concentrations.The GLUT4/muscle-fat glucose transporter is one member of the facilitative glucose transporter super-gene family that is specifically expressed in muscle and adipose tissues (1, 2). In contrast to the other glucose transporter isoforms, GLUT4 contains specific amino acid targeting sequences (3-5) responsible for its localization to unique intracellular vesicular compartments found in adipocytes and muscle cells (6)(7)(8)(9)(10)(11). In response to acute insulin stimulation, these preformed GLUT4-containing vesicles rapidly translocate to the plasma membrane in a GTP-dependent process, resulting in a large increase in plasma membrane-associated GLUT4 protein (9-17).In contrast to this acute pathway of insulin action, catabolic states such as fasting and non-insulin-dependent diabetes are directly associated with a marked resistance of adipose and muscle tissue to insulin-stimulated glucose uptake (18)(19)(20). Recently, several studies have suggested that a decrease in GLUT4 expression may be the initial cause of insulin resistance in adipose tissue, which contributes to the maintenance ofinsulin resistance in muscle (21)(22)(23)(24)(25)(26)(27)(28)(29). Since the pathophysiological mechanisms responsible for insulin resistance are poorly understood, we have recently generated transgenic mice expressing high levels of the human GLUT4The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indi...