Transport of protein between cytoplasmic membranes of fused cells: correspondence to processes reconstituted in a cell-free system.

Abstract

Mixed monolayers containing vesicular stomatitis virus-infected Chinese hamster ovary clone 15B cells (lacking UDP-N-acetylglucosamine transferase I, a Golgi enzyme) and uninfected wild-type Chinese hamster ovary cells were formed . Extensive cell fusion occurs after the monolayer is exposed to a pH of 5.0. The vesicular stomatitis virus encoded membrane glycoprotein (G protein) resident in the rough endoplasmic reticulum (labeled with ["S]methionine) or Golgi complex (labeled with [3 H]palmitate) of 15B cells at the time of fusion can reach Golgi complexes from wild-type cells after fusion ; G protein present in the plasma membrane cannot. Transfer to wild-type Golgi complexes is monitored by the conversion of G protein to an endoglycosidase H-resistant form upon arrival, and also demonstrated by immunofluorescence microscopy. G protein in the Golgi complex of the 15B cells at the time of fusion exhibits properties vis à vis its transfer to an exogenous Golgi population identical to those found earlier in a cell-free system (Fries, E., and J . E . Rothman . 1981 . J. Cel l Biol., 90: 697-704) . Specifically, pulse-chase experiments using the in vivo fusion and in vitro assays reveal the same two populations of G protein in the Golgi complex . The first population, consisting of G protein molecules that have just received their fatty acid, can transfer to a second Golgi population in vivo and in vitro. The second population, entered by G protein -5 min after its acylation, is unavailable for this transfer, in vivo and in vitro. Presumably, this second population consists of those G-protein molecules that had already been transferred between compartments within the 15B Golgi population, in an equivalent process before cell fusion or homogenization for in vitro assays . Evidently, the same compartment boundary in the Golgi complex is detected by these two measurements . The surprisingly facile process of glycoprotein transit between Golgi stacks that occurs in vivo may therefore be retained in vitro, providing a basis for the cell-free system .

A Novel Stress-induced EDEM Variant Regulating Endoplasmic Reticulum-associated Glycoprotein Degradation

“…The mobility shift was the same just after synthesis when the proteins are in the ER and all N-glycans are Endo H-sensitive and 2 h after synthesis, as a clear indication that all glycans had maintained Endo H sensitivity (Fig. 1B) and that the proteins were not transported to the Golgi compartment (29). The localization of EDEM and EDEM2 in the ER was also confirmed by immunofluorescence (data not shown).…”

Intercompartmental transport in the Golgi complex is a dissociative process: facile transfer of membrane protein between two Golgi populations.

“…The CHO mutant Lec2 and its wild-type parent W5 were generously provided by Pamela Stanley (Albert Einstein College of Medicine, Bronx, NY) . All were grown in monolayer in Eagle's minimum essential medium as described (21) .…”

“…Preparation of Mixed Monolayers: These were formed by exactly the procedures described in the preceding article (21), except the CHO cell lines employed were different. For one set of experiments, the infected cells were of clone 13 and the uninfected cells were of clone 15B .…”

“…The question is, can G protein, labeled in its GIcNAc residues in the clone 13 Golgi complex, be transported to another Golgi population where it will be further glycosylated with Gal and possibly other sugars? The preceding paper (21) shows that two Golgi populations derived from two types of CHO cells remain distinct after cell fusion.…”

“…To ascertain whether G protein present in the Golgi complex of one cell can move to the Golgi complex of another after fusion, mixed monolayers were formed by the procedure of the preceding paper (21). These consisted of VSV-infected CHO clone 13 cells and uninfected CHO clone 15B cells (schematized in Fig.…”