Showing papers by "David D. Sabatini published in 1985"

Polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of Madin-Darby canine kidney cells infected with temperature-sensitive viruses.

Abstract: The intracellular route followed by viral envelope glycoproteins in polarized Madin-Darby canine kidney cells was studied by using temperature-sensitive mutants of vesicular stomatitis virus (VSV) and influenza, in which, at the nonpermissive temperature (395 degrees C), the newly synthesized glycoproteins (G proteins) and hemagglutinin (HA), respectively, are not transported out of the endoplasmic reticulum After infection with VSV and incubation at 395 degrees C for 4-5 h, synchronous transfer of G protein to the plasma membrane was initiated by shifting to the permissive temperature (325 degrees C) Immunoelectron microscopy showed that under these conditions the protein moved to the Golgi apparatus and from there directly to a region of the lateral plasma membrane near this organelle G protein then seemed to diffuse progressively to basal regions of the cell surface and, only after it had accumulated in the basolateral domain, it began to appear on the apical surface near the intercellular junctions The results of these experiments indicate that the VSV G protein must be sorted before its arrival at the cell surface, and suggest that passage to the apical domain occurs only late in infection when tight junctions are no longer an effective barrier In complementary experiments, using the temperature-sensitive mutant of influenza, cultures were first shifted from the nonpermissive temperature (395 degrees C) to 185 degrees C, to allow entrance of the glycoprotein into the Golgi apparatus (see Matlin, KS, and K Simons, 1983, Cell, 34:233-243) Under these conditions HA accumulated in Golgi stacks and vesicles but did not reach the plasma membrane When the temperature was subsequently shifted to 325 degrees C, HA rapidly appeared in discrete regions of the apical surface near, and often directly above, the Golgi elements, and later diffused throughout this surface To ensure that the anti-HA antibodies had access to lateral domains, monolayers were treated with a hypertonic medium to dilate the intercellular spaces Some labeling was then observed in the lateral plasma membranes soon after the shift, but this never increased beyond 10 gold particle/micron, whereas characteristic densities of labeling in apical surfaces soon became much higher (approximately 10 particles/micron) Our results suggest that the bulk of HA follows a direct pathway leading from the Golgi to regions of the apical surface close to trans-Golgi cisternae

Biosynthesis and intracellular sorting of growth hormone-viral envelope glycoprotein hybrids.

Abstract: Various aspects of the biogenetic mechanisms that are involved in the insertion of nascent plasma membrane proteins into the endoplasmic reticulum (ER) membrane and their subsequent distribution through the cell have been investigated. For these studies chimeric genes that encode hybrid proteins containing carboxy-terminal portions of the influenza virus hemagglutinin (154 amino acids) or the vesicular stomatitis virus envelope glycoprotein (G) (60 amino acids) linked to the carboxy terminus of a nearly complete secretory polypeptide, growth hormone (GH), were used. In in vitro transcription-translation experiments, it was found that the insertion signal in the GH portion of the chimeras led to incorporation of the membrane protein segments into the ER membrane. Effectively, GH became part of the luminal segment of membrane proteins of which only very small segments, corresponding to the cytoplasmic portions of the G or HA proteins, remained exposed on the surface of the microsomes. When the chimeric genes were expressed in transfected cells, the products, as expected, failed to be secreted and remained cell-associated. These results support the assignment of a halt transfer role to segments of the membrane polypeptides that include their transmembrane portions. The hybrid polypeptide containing the carboxy-terminal portion of HA linked to GH accumulated in a juxtanuclear region of the cytoplasm within modified ER cisternae, closely apposed to the Golgi apparatus. The location and appearance of these cisternae suggested that they represent overdeveloped transitional ER elements and thus may correspond to a natural way station between the ER and the Golgi apparatus, in which further transfer of the artificial molecules is halted. The GH-G hybrid could only be detected in transfected cells treated with chloroquine, a drug that led to its accumulation in the membranes of endosome or lysosome-like cytoplasmic vesicles. Although the possibility that the chimeric protein entered such vesicles directly from the Golgi apparatus cannot be ruled out, it appears more likely that it was first transferred to the cell surface and was then internalized by endocytosis.