An essential role for the phosphatidylinositol transfer protein in the scission of coatomer-coated vesicles from the trans-Golgi network.

We identified the phosphatidylinositol transfer protein (PITP) as being responsible for a powerful latent, nucleotide-independent, Golgi-vesiculating activity that is present in the cytosol but is only manifested as an uncontrolled activity in a cytosolic protein subfraction, in which it is separated from regulatory components that appear to normally limit its action to the scission of COPI-coated buds from trans-Golgi network membranes. A specific anti-PITP antibody that recognizes the two mammalian PITP isoforms fully inhibited the capacity of the cytosol to support normal vesicle generation as well as the uncontrolled vesiculating activity manifested by the cytosolic protein subfraction. The phosphatidylinositol- (PI) loaded form of the yeast PITP, Sec14p, but not the phosphatidylcholine- (PC) loaded form of the protein, was capable of substituting for the cytosolic subfraction in promoting the scission of coated buds from the trans-Golgi network. At higher concentration, however, Sec14p, when loaded with PI, but not with PC or phosphatidylglycerol, caused by itself an indiscriminate vesiculation of uncoated Golgi membranes that could be suppressed by PC-Sec14p, which also suppresses the uncontrolled vesiculation caused by the cytosolic subfraction. We propose that, by delivering PI to specific sites in the Golgi membrane near the necks of coated buds, PITP induces local changes in the organization of the lipid bilayer, possibly involving PI metabolites, that triggers the fusion of the ectoplasmic faces of the Golgi membrane necessary for the scission of COPI-coated vesicles.

Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells.

Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm.

[Use of cultured, virus-infected cells to study the biogenesis of polarity of epithelial cells]. / Utilisation de cultures de cellules infectées par des virus pour l'étude de la biogénèse de la polarité des cellules épithéliales.

The major characteristic of the eucaryote cell is the presence of specialized organelles in which macromolecular components responsible for various subcellular functions are segregated. The membranes of these organelles serve not only as divisions between the various cytoplasmic compartments, but also provide scaffolding within which the macromolecular complexes of the organelle assemble and become functionally integrated. It is obvious that because of the degree of complexity resulting from the existence of numerous compartments and membrane systems, the development of a genetic programme in a eucaryote cell requires not only the transcription of specific genes and translation in the cytoplasma of the resultant messenger RNA, but also the activity of mechanisms which ensure that each polypeptide reaches the site of its function, which may be in the cytosol, in a membrane, or in the luminal cavity of an organelle. In the special case of membrane proteins, such mechanisms must result not only in the specific distribution of polypeptides newly synthesized in the various types of cell membrane, but also the arrangement of them required in the lipid bi-layer necessary for their normal function.

Sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells.

Previous studies (Rindler, M. J., I. E., Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100: 136-151; Rindler, M. J., I. E. Ivanov, H. Plesken, E. J. Rodriguez-Boulan, and D. D. Sabatini, 1984, J. Cell Biol., 98: 1304-1319) have demonstrated that in polarized Madin-Darby canine kidney cells infected with vesicular stomatitis virus (VSV) or influenza virus the viral envelope glycoproteins G and HA are segregated to the basolateral and apical plasma membrane domains, respectively, where budding of the corresponding viruses takes place. Furthermore, it has been shown that this segregation of the glycoproteins reflects the polarized delivery of the newly synthesized polypeptides to each surface domain. In transfection experiments using eukaryotic expression plasmids that contain cDNAs encoding the viral glycoproteins, it is now shown that even in the absence of other viral components, both proteins are effectively segregated to the appropriate cell surface domain. In transfected cells, the HA glycoprotein was almost exclusively localized in the apical cell surface, whereas the G protein, although preferentially localized in the basolateral domains, was also present in lower amounts, in the apical surfaces of many cells. Using transfected and infected cells, it was demonstrated that, after reaching the cell surface, the G protein, but not the HA protein, undergoes interiorization by endocytosis. Thus, in the presence of chloroquine, a drug that blocks return of interiorized plasma membrane proteins to the cell surface, the G protein was quantitatively trapped in endosome- or lysosome-like vesicles. The sequestration of G was a rapid process that was completed in many cells by 1-2 h after chloroquine treatment. The fact that in transfected cells the surface content of G protein was not noticeably reduced during a 5-h incubation with cycloheximide, a protein synthesis inhibitor that did not prevent the effect of chloroquine, implies that normally, G protein molecules are not only interiorized but are also recycled to the cell surface.

Secretion of endogenous and exogenous proteins from polarized MDCK cell monolayers.

Confluent monolayers of MDCK (Madin-Darby canine kidney) cells provide a widely used system to study the biogenesis of epithelial cell polarity. We now report that these cells are also capable of the vectorial constitutive secretion of a major endogenous product, a glycoprotein of 81 kDa, which is released into the medium from the apical surface within 30 min of its synthesis. This release represents a bona fide exocytotic secretory process and is not the result of proteolytic cleavage of a plasma membrane-associated precursor since, in cells treated with chloroquine, a protein indistinguishable from the mature secretory product accumulated intracellularly. In contrast to the vectorial secretion of the endogenous product, a variety of exogenous exocrine and endocrine proteins synthesized in MDCK cells transfected with the corresponding genes were secreted from both the apical and basolateral surfaces. These included proteins such as rat growth hormone, chicken oviduct lysozyme, bovine gastric prochymosin, and rat salivary gland alpha 2u-globulin, which in their cells of origin are secreted via a regulated pathway, as well as the liver form of the alpha 2u-globulin and the immunoglobulin kappa chain, which are normally released constitutively. These results demonstrate the existence of secretory pathways that lead to both surfaces of MDCK cells and are accessible to the foreign secretory products. They are consistent with the operation of a sorting mechanism in which the polarized secretion of the endogenous product is effected through the recognition of signals that prevent its random distribution within the fluid phase in the cellular endomembrane system.

Intracellular glycosylation of vitellogenin in the liver of estrogen-stimulated Xenopus laevis.

Pulse-chase experiments measuring the rates of incorporation of radiolabeled glucosamine and galactose into intracellular vitellogenin show that glycosylation of this multicomponent protein occurs in a Golgi-enriched fraction isolated from homogenized liver slices. No apparent role of the rough endoplasmic reticulum in this process was demonstrable. Kinetics of the intracellular translocation of glycosylated vitellogenin indicate that the galactosylated intermediate is secreted more rapidly than the glucosamine-labeled precursor. This was corroborated by measuring the rates of accumulation of various pulse-labeled forms of vitellogenin in the chase medium. In addition, a negligible amount of mannose was incorporated into intracellular or secreted vitellogenin. The antibiotic tunicamycin was shown to inhibit [3H] glucosamine incorporation into microsomal vitellogenin by 70%, without any significant effect on the synthesis of the protein backbone. In addition, nonglycosylated vitellogenin showed normal secretion kinetics. After suitable pretreatment with the antibiotic followed by a labeling period in tunicamycin-free medium, mannose was still not incorporated into vitellogenin, whereas glucosamine behaved in a typical manner. In contrast to this finding, gas-liquid chromatography of the alditol acetate derivatives of the neutral hexoses of vitellogenin showed that mannose was indeed a major component of the vitellogenin oligosaccharide side chain. These preliminary results indicate that the oligosaccharide component of vitellogenin in Xenopus laevis is a "complex" type of carbohydrate unit which is linked via an N-glycosidic bond between an asparagine residue and N-acetylglucosamine. With respect to the subcellular localization of glycoprotein assembly in Xenopus liver, there is a significant departure from currently accepted models of glycoprotein synthesis.

Intracellular phosphorylation of vitellogenin in the liver of estrogen-stimulated Xenopus laevis.

A procedure was developed for the preparation of rough and smooth microsomes from small quantities of liver obtained from estrogen-stimulated Xenopus laevis females. The separation involves th centrifugation of a postmitochondrial supernatant over a CsCl-containing, discontinuous sucrose gradient. Morphological, biochemical, and enzymatic characterization of these fractions indicates that excellent separation of rough microsomes from smooth microsomes is achieved. In addition, pulse-chase experiments in which (5-min) pulses with [3H]leucine are used demonstrate that rough and smooth microsomes each exhibit the predictable patterns of incorporation characteristic of secretory protein synthesis and intracellular translocation. This procedure was combined with suitable incubation conditions for pulse-chase experiments which demonstrate the subcellular sites of vitellogenin phosphorylation. The data presented indicate that approximately 70% of the phosphate residues are covalently attached to vitellogenin during its intracellular translocation through the smooth microsomes, while the rough microsomes can account for the remainder of the total incorporated phosphate. This is further supported by the analysis of newly synthesized and assembled [3H,32P]vitellogenin on sodium dodecyl sulfate-polyacrylamide gels and measurements of protein kinase activity in microsomal subfractions.