A 58-kDa resident protein of the cis Golgi cisterna is not terminally glycosylated.

203

284

Abstract. The glycoside digitonin was used to selectively permeabilize the plasma membrane exposing functionally and morphologically intact ER and Golgi compartments. Permeabilized cells efficiently transported vesicular stomatitis virus glycoprotein (VSV-G) through sealed, membrane-bound compartments in an ATP and cytosol dependent fashion. Transport was vectorial. VSV-G protein was first transported to punctate structures which colocalized with p58 (a putative marker for peripheral punctate pre-Golgi intermediates and the cis-Golgi network) before delivery to the medial Golgi compartments containing tx-l,2-mannosidase II and processing of VSV-G to endoglycosidase H resistant forms. Exit from the ER was inhibited by an antibody recognizing the carboxyl-terminus of VSV-G. In contrast, VSV-G protein colocalized with p58 in the absence of Ca 2+ or the presence of an antibody which inhibits the transport component NSF (SEC18). These studies demonstrate that digitonin permeabilized cells can be used to efficiently reconstitute the early secretory pathway in vitro, allowing a direct comparison of the morphological and biochemical events involved in vesicular tafficking, and identifying a key role for the p58 containing compartment in ER to Golgi transport.

Section: Transport In Vitro Occurs Through the P58 Containing Compartmentsupporting

confidence: 88%

Section: Transport In Vitro Occurs Through the P58 Containing Compartmentmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Morphological analysis of protein transport from the ER to Golgi membranes in digitonin-permeabilized cells: role of the P58 containing compartment.

Plutner

Davidson

Saraste

et al. 1992

203

284

“…The budding compartment labeled with p58, a well characterized marker of the intermediate compartment (Saraste and Svensson, 1991). Significantly, the O-linked oligosaccharides of this protein have been shown to contain terminal GalNAc, consistent with its localization in the intermediate compartment (Hendricks et al, 1991). It also labeled for the small GTPase rab2, which has been localized to the intermediate compartment (Chavrier et al, 1990).…”

Section: The Mhv Budding Compartment Is Continuous With the Rough Er And Labels For Both Er And Intermediate Compartment Markerssupporting

confidence: 60%

Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step

Krijnse‐Locker

Ericsson

Rottier

et al. 1994

318

256

Abstract. Mouse hepatitis coronavirus (MHV) buds into pleomorphic membrane structures with features expected of the intermediate compartment between the ER and the Golgi complex. Here, we characterize the MHV budding compartment in more detail in mouse L cells using streptolysin O (SLO) permeabilization which allowed us to better visualize the membrane structures at the ER-Golgi boundary. The MHV budding compartment shares membrane continuities with the rough ER as well as with cisternal elements on one side of the Golgi stack. It also labeled with p58 and rab2, two markers of the intermediate compartment, and with PDI, usually considered to be a marker of the rough ER. The membranes of the budding compartment, as well as the budding virions themselves, but not the rough ER, labeled with the N-acetyl-galactosamine (GalNAc)-specific lectin Helix pomatia. When the SLO-permeabilized cells were treated with guanosine 5'-(3-O-thio)triphosphate (GTP',/S), the budding compartment accumulated a large number of ~-cop-containing buds and vesicular profiles.Complementary biochemical experiments were carried out to determine whether vesicular transport was required for the newly synthesized M protein, that contains only O-linked oligosaccharides, to acquire first, GalNAc and second, the Golgi modifications galactose and sialic acid. The results from both in vivo studies and from the use of SLO-permeabilized cells showed that, while GalNAc addition occurred under conditions which block vesicular transport, both cytosol and ATP were prerequisites for the M protein oligosaccharides to acquire Golgi modifications. Collectively, our data argue that transport from the rough ER to the Golgi complex requires only one vesicular transport step and that the intermediate compartment is a specialized domain of the endoplasmatic reticulum that extends to the first cisterna on the cis side of the Golgi stack.THOUGH the boundary region between the ER and the Golgi complex has been extensively investigated during the past few years, many questions remain about the organization of this part of the cell. Foremost among these is whether a separate intermediate compartment exists or whether this structure is functionally continuous with the ER. The available evidence strongly suggests that both in mammalian cells (Beckers et al., 1990) and in yeast (Rexach and Schekman, 1991) at least one vesicular transport step exists between the rough ER and the Golgi complex. A separate intermediate compartment, as has been postulated by several authors (Warren, 1987;Pelham, 1989; MeUman and Simons, 1992) would necessitate two distinct vesicular transport steps, one from the ER to the intermediate compartment and the other from this site to Golgi. In contrast, if the intermediate compartment is continuous with the ER, transport to the Golgi complex would require a single vesicular transport step.

“…These data have led to the view that enzymes involved in the three stages of construction are physically separated in cisternae that are ordered in a stack. There have been occasional reports of Golgi residents occupying more than one group of cisternae (Novikoff et al, 1983;Roth et al, 1986;Hendricks et al, 1991) but they have generally been regarded as exceptions to the rule.…”

mentioning

confidence: 99%

Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells.

Nilsson

Pypaert

Hoe

et al. 1993

213

154

Abstract. Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme,/31,4 gaiactosyltransferase (GaiT) and the medial enzyme, N-acetylglucosaminylransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myctagged version of the endogenous protein. GAIT was found in the trans-cisterna and trans-Golgi network but, contrary to expectation, NAGT I was found both in the medial-and trans-cisternae, overlapping the distribution of GAIT. About one third of the NAGT I and half of the GaIT were found in the shared, transcisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.T HE construction of N-linked, bi-antennary oligosaccharides in the Golgi apparatus is a three-stage process.The first stage, thought to take place in the cis-Golgi network (CGN)l/cis-cisternae, continues the trimming of mannose residues started in the ER. The second stage, in medial-cisternae, involves addition of N-acetylglucosamine, the removal of a further two mannose residues and the addition of another N-acetylglucosamine. Fucose may also be added at this stage. The third and last stage, in the transcisternae and trans-Golgi network (TGN), involves addition of galactose followed by sialic acid (for review see Kornfeld and Kornfeld, 1985).These activities have been assigned to the different cisternae and networks based on localiTation of the appropriate enzymes and their products (for reviews see Rothman, 1985, andRoth, 1987). The first stage enzyme, mannosidase I, is thought to reside in the CGN/cis-cisternae because it acts on oligosaccharides attached to proteins that have just left the ER (Balch et al., 1986) and before the second stage enzyme, N-acetylglucosaminyltransferase I (NAGT I), which has been immunolocalized to medialcisternae . Other second stage enzymes (mannosidase II and NAGT H) cofractionate with NAGT I on sucrose gradients and, since they can be partially separated from third-stage enzymes (/31,4 galactosyltransferase [GAIT] and ot2,6 sialyltransferase [SialylT]) on the same gradients (Dunphy et al., 1981; Dunphy and Rothman, Tommy Nilsson and Marc Pypaert contributed equally to this work.