Cell type-specific post-Golgi apparatus localization of a "resident" endoplasmic reticulum glycoprotein, glucosidase II.

Abstract

Abstract. Glucosidase II, an asparagine-linked oligosaccharide processing enzyme, is a resident glycoprotein of the endoplasmic reticulum. In kidney tubular cells, in contrast to previous findings on hepatocytes, we found by light and electron microscopy immunoreactivity for glucosidase II predominantly in post-Golgi apparatus structures. The majority of immunolabel was in endocytotic structures beneath the plasma membrane. Immunoprecipitation confirmed presence of the glucosidase II subunit in purified brush border preparations. Kidney glucosidase II conmined species carrying endo H-sensitive, high mannose as well as endo H-resistant oligosaccharide chains. Some species of glucosidase II contained sialic acid. The sialylated species were enzymatically active. This study demonstrates than an enzyme presumed to be a resident of the endoplasmic reticulum may show alternative localizations in some cell types.N '-glycosylated glycoproteins acquire their oligosaccharide chains by en bloc transfer of the lipid-linked precursor Glc3Man9GlcNAc2 to the nascent polypeptide. The oligosaccharide precursors are then modified by glycosidases and glycosyltransferases to generate high marmose-, hybrid-, or complex-type structures. The processing starts with the removal of glucose residues by glucosidases (Hirschberg and Snider, 1987). Glucosidase I removes rapidly (tt/2 <1 min) the terminal od,2-1inked glucose residue (Hubbard and Robbins, 1979). Glucosidase II hydrolyzes the two inner od,3-1inked glucose residues (Grinna and Robbins, 1979;Burns and Touster, 1982). The outer cd,3-linked glucose residue is hydrolyzed more rapidly (t~ --5 rain) than the inner one (after •20-30 min) (Hubbard and Robbins, 1979). In hepatocytes, glucosidase II is a high mannose-type glycoprotein and was localized to the rough and smooth endoplasmic reticulum by biochemical fractionation procedures (Grinna and Robbins, 1979;Brada and Dubach, 1984) and by immunoelectron microscopy (Lucocq et al., 1986). Golgi apparatus cisternae were free of immunolabel for glucosidase II although subcellular fractions of Golgi apparatus contained small amounts of the enzyme which almost certainly represented contamination by the endoplasmic reticulum. The data on the biosynthesis of glucosidase II obtained in a rat hepatoma cell line are in agreement with the above results (Strous et al., 1987). The enzyme subunit was found to be an endo H-sensitive, 100-kD glycoprotein that remained unchanged over long chase periods. The halflife of glucosidase II was not altered by monensin treatment, further supporting the notion that glucosidase II does not enter the Golgi apparatus in hepatocytes. Collectively, these data demonstrated that, in hepatocytes, glucosidase II is a resident glycoprotein of the endoplasmic reticulum which is retained therein by an unknown mechanism.In the present study we have investigated the distribution of glucosidase II in pig kidney. The examination of kidney tubular cells revealed unexpected findings. Our biochemical and immunocytochemical data ...

Subcellular localization of the UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation reaction in the submaxillary gland.

“…Others, using the Helix pomatia lectin and monoclonal anti-Tn antibodies, reported the presence of GalNAc residues in limited portions of the rough ER (6,7). It is possible that the transferase is localized differently in different cell types (13,36,37) but the primary chondrocyte cultures (6) in which it appeared that GalNAc residues were in the ER may well have been structurally altered as a consequence of the prolonged culture conditions (6). Indeed, the regions of the ER that appeared to contain GatNAc residues were structurally different from the ER in healthy cells.…”

Cysts of PRKCSH mutated polycystic liver disease patients lack hepatocystin but express Sec63p

“…Hepatocystin was found in the membrane fraction of the human kidney cell line HEK293 when co-expressed with the ion channel TRPV5, and hepatocystin translocates to the nucleus when MCF-7 cells are treated with Wbroblast growth factor 1 (Forough et al 2003;Gkika et al 2004). Furthermore, in pig liver, hepatocystin's partner glucosidase II is localized in both rough and smooth ER (Lucocq et al 1986), but in pig kidney it is also localized in the Golgi apparatus (Brada et al 1990). Consequently, there is little consensus on the cellular localization of hepatocystin and it is unclear in which cell compartment of human liver cells hepatocystin executes its function.…”

Golgi Apparatus Immunolocalization of Endomannosidase Suggests Post-Endoplasmic Reticulum Glucose Trimming: Implications for Quality Control

“…In the present study, an IgG fraction prepared by protein A-Sepharose chromatography from this antiserum was used. Furthermore, polyclonal rabbit antibodies against pig and rat glucosidase II (Lucocq et al, 1986;Brada et al, 1990), calreticulin (Peter et al, 1992; kindly provided by Dr. H. D. Sö ling, Gö ttingen, Germany), rat p58 (Saraste and Svensson, 1991; affinity-purified and kindly provided by Dr. J. Saraste, University of Bergen, Norway), and Golgi mannosidase II (Velasco et al, 1993; kindly provided by Dr. K. Moremen, University of Georgia, Athens, GA) were used. A mouse monoclonal anti-Golgi mannosidase II antibody (ascites form) was purchased from Babco (Richmond, CA).…”