Abstract
During short incubations of a Golgi apparatus-enriched subcellular fraction from rat liver with UDP-[3H]GlcNAc, label is efficiently transferred to endogenous acceptors. Most of the macromolecular radioactivity is specifically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that it is mainly associated with N-linked oligosaccharides. The glycoprotein acceptors are resistant to proteases unless detergent is added in amounts greater than the critical micellar concentration. This shows that the acceptors are within the lumen of intact compartments, which have the correct topological orientation expected for the Golgi apparatus in intact cells. Structural characterization of the radiolabeled N-linked oligosaccharides shows a variety of distinct neutral and anionic species. The neutral chains include bi-, tri-, and tetra-antennary molecules with terminal beta-[3H] GlcNAc residues. In vitro sialylation shows that some of the tetra-antennary chains have beta 1,3-linked Gal residues on their unlabeled antennae. An unknown modification appears to block the action of beta-galactosidase on these galactosylated oligosaccharides. Chasing the labeling reaction with a mixtures of UDP-Gal, CMP-Neu5Ac, and adenosine 3'-phosphate,5'-phosphosulfate causes an increase in the percent of radiolabeled anionic oligosaccharides. Most of the negative charge is due to sialic acid (Sia), and some appears to be in phosphodiester-linked [3H]GlcNAc. The sialylated oligosaccharides are a mixture of bi-, tri-, and tetra-antennary species with 1-3-Sia residues, and some of the [3H]GlcNAc residues are directly covered with unlabeled Gal and Sia residues. This in vitro approach should recapitulate reactions that occur in the biosynthesis of N-linked oligosaccharides in the Golgi apparatus of the intact cell. Since the conditions during labeling do not permit inter-compartmental transport, the oligosaccharides produced should represent the biosynthetic capabilities of individual Golgi compartments. Evidence is presented for a functional association of GlcNAc transferases I, II, and alpha-mannosidase II, with separation from GlcNAc transferase IV and/or V. The structures also indicate co-compartmentalization of several GlcNAc transferase(s) with beta-galactosyltransferase(s) and sialyltransferase(s). The compartmental organization of the Golgi apparatus is discussed in light of these findings.
Highlights
The sugar nucleotide transporters are known to be capable of concentrating the nucleotides 20-50-fold in the Golgi apparatus [12, 13]
We have presented here a novel approach to studying the biosynthetic function and organization of the Golgi apparatus
The approach originates from previously well known studies of the uptake and concentrationof sugar nucleotides by intact Golgi compartments [12,13, 54, 55],andothers in which radioactive sugar nucleotides were added to microsomal preparations, with analysis of labeled products [17, 56, 57]
Summary
Characterization of the Golgi-enriched Fractwn-A subcellular fractionenriched in theGolgi apparatus (Golgi-enriched fraction) was prepared from rat liver as described under “Experimental Procedures.” The procedure is essentially as previously described [27] except that the final salt wash and pelleting steps were omitted. While Triton X-100 at high concentrations can nonspecifically inhibit acid precipitation, thiseffect was not apparent at 0.05%, where incorporation was already reduced by 89% These results suggest that detergent disrupts the Golgi membrane and abolishes a concentration gradient of UDP-[3H]GlcNAc, and indicate that the transfer reaction is taking place in intact membranebound compartments. Liquots of a freshly isolated Golgienriched fraction were preincubated with increasing concentrations of Triton X-100, incubated with UDP-[3H]GlcNAc,and theradioactivity incorporated into macromoleculeswas determined as described under “Experimental Procedures.”. Initial experiments using Golgienriched fractions incubated with UDP-[3H]GlcNAcshowed that MnClzcaused a dose-dependent increase in incorporation of label into macromolecules, and that the addition of other sugar nucleotides could alterthe N-linked oligosaccharide structures produced (data notshown). The released oligosacchacan transfer [3H]GlcNAcfrom UDP-[3H]GlcNAc to macro- rides were separated from macromolecules by rechromatogmolecular acceptors Thelabeling of such Golgi-enriched preparations should reflect the glycosylation reactions that normally occur in vivo. The radiolabeled N-linked oligosaccharides were fractionated by ConA-Sepharose as described under “Experimental Procedures.” The percent of radiolabel that was either unretained or eluted from the column is shown
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