Carlos B. Hirschberg

Topography of glycosylation reactions in the endoplasmic reticulum.

A variety of distinct protein glycosylation reactions occur in the endoplasmic reticulum (ER) of eukaryotic cells. In some instances, both the proteins to be glycosylated and the precursor sugar donors must be translocated across the membrane from the cytoplasm to the lumen of the ER. Elucidation of the individual steps in each of the glycosylation pathways has revealed the topographic complexity of these reactions.

Transporters of nucleotide sugars, nucleotide sulfate and ATP in the Golgi apparatus

Proteins and glycolipids are glycosylated, sulfated and phosphorylated in the lumen of the Golgi apparatus. The nucleotide substrates of these reactions must first be translocated from the cytosol into the Golgi lumen by specific transporters (antiporters). These are hydrophobic, transmembrane spanning proteins that appear to regulate post-translational modifications in the Golgi lumen.

The glycolipids and phospholipids of Sindbis virus and their relation to the lipids of the host cell plasma membrane

Abstract Sindbis virus grown in chick embryo fibroblasts contains four sialoglycolipids which are apparently the same as those of the host cell. The principal glycolipid is hematoside. The phospholipid composition of Sindbis virus is similar to that of a plasma membrane preparation obtained from chick embryo fibroblasts. These results indicate that the proteins of the virion incorporate a nonspecific population of host cell plasma membrane phospholipids and glycolipids during viral morphogenesis.

A Mutant Yeast Deficient in Golgi Transport of Uridine Diphosphate N-Acetylglucosamine

Mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they have terminal α12-linked N-acetylglucosamine and lack mannose phosphate. In a previous study, Douglas and Ballou (Douglas, R. K., and Ballou, C. E. (1982) Biochemistry 21, 1561-1570) characterized a mutant, mnn2-2, which lacked terminal N-acetylglucosamine in its mannoproteins. The mutant had normal levels of N-acetylglucosaminyltransferase activity, and the partially purified enzyme from wild-type and mutant cells had the same apparent size, heat stability, affinity for substrates, metal requirement, and subcellular location. No qualitative or quantitative differences were found between mutant and wild-type cells in endogenous mannan acceptors and pools of UDP-GlcNAc. Chitin was synthesized at similar rates in wild-type and mutant cells, and the latter did not have a soluble inhibitor of the N-acetylglucosaminyltransferase or a hexosaminidase that could remove N-acetylglucosamine from mannoproteins. Together, the above observations led Douglas and Ballou ((1982) Biochemistry 21, 1561-1570) to postulate that the mutant might have a defect in compartmentation of substrates involved in the biosynthesis of mannoproteins. We determined whether the above mutant phenotype is the result of defective transport of UDP-GlcNAc into Golgi vesicles from K. lactis. Golgi vesicles which were sealed and of the same membrane topographical orientation as in vivo were isolated from wild-type and mnn2-2 mutant cells and incubated with UDP-GlcNAc in an assay in vitro. The initial rate of transport of UDP-GlcNAc into Golgi vesicles from wild-type cells was temperature dependent, saturable with an apparent K of 5.5 μM and a V of 8.2 pmol/mg of protein/3 min. No transport of UDP-GlcNAc was detected into Golgi vesicles from mutant cells. However, Golgi vesicles from both cells translocated GDP-mannose at comparable velocities, indicating that the above transport defect is specific. In addition to the above defect in mannoproteins, mutant cells were also deficient in the biosynthesis of glucosamine containing lipids.

Identification, Purification, and Characterization of the Rat Liver Golgi Membrane ATP Transporter

Phosphorylation of secretory and integral membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi membrane has been previously demonstrated in vitro using intact Golgi membrane vesicles from rat liver and mammary gland. We have now identified and purified the rat liver Golgi membrane ATP transporter. The transporter was purified to apparent homogeneity by a combination of conventional ion exchange, dye color, and affinity chromatography. An ∼70,000-fold purification (2% yield) was achieved starting from crude rat liver Golgi membranes. A protein with an apparent molecular mass of 60 kDa was identified as the putative transporter by a combination of column chromatography, photoaffinity labeling with an analog of ATP, and native functional size determination on a glycerol gradient. The purified transporter appears to exist as a homodimer within the Golgi membrane, and when reconstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3′-phosphate 5′-phosphosulfate transport. The transport activity was saturable with an apparentK m very similar to that of intact Golgi vesicles.

Transporters of nucleotide sugars, nucleotide sulfate and ATP in the Golgi apparatus membrane: Where next?

Recently the UDP-GlcNAc, CMP-sialic acid and GDPmannose transporters from the Golgi apparatus membrane were cloned (Abeijon et al., 1996; Eckhardt et al., 1996; Ma et al., 1997). These transporters, as well as those for other nucleotide sugars, PAPS and ATP, are required for these nucleotide derivatives to reach to Golgi lumen from the cytosol and serve as substrates in glycosylation, sulfation, and phosphorylation of glycoproteins, proteoglycans, and glycolipids. Previously, biochemical and genetic evidence demonstrated that these transporters are highly specific for solute transport (Hirschberg, 1996; Hirschberg and Snider, 1987), are often organelle specific (Hirschberg, 1996), appear to play a regulatory role in the biosynthesis of Golgi lumenal macromolecules (Abeijon et al., 1993; Toma et al., 1996), and use antiporters with the corresponding nucleoside phosphate as the mechanism for concentration of solutes in the Golgi lumen (Hirschberg and Snider, 1987; Milla and Hirschberg, 1989; Waldman and Rudnick, 1990; Abeijon et al., 1993; Berninsone et al., 1994). The aim of this article is not to provide a comprehensive review of this topic, which has been done recently (Hirschberg, 1996), but to highlight some important unanswered questions and new avenues of experimentation of this topic.

Inhibition of terminal N- and O-glycosylation specific for herpesvirus-infected cells: mechanism of an inhibitor of sugar nucleotide transport across Golgi membranes

The nucleoside analog (E)-5-(2-bromovinyl)-2-deoxyuridine (BVdU) inhibited the Golgi-associated terminal glycosylation in herpes simplex virus type 1- and type 2-infected cells, specifically incorporation of galactose and sialic acid into N-linked oligosaccharides, and incorporation of sialic acid and, to a lesser extent, of galactose into O-linked oligo saccharides. This resulted in formation of viral glycoproteins with terminal GlcNAc and Fuc in N-linked oligosaccharides and terminal O-linked GalNAc. Inhibition of formation of UDP-hexoses and of acceptor glycoprotein synthesis and inhibition of cellular transport of viral glycoproteins were not observed. No evidence for the formation of a sugar nucleotide analog of BVdU was obtained. Inhibition required phosphorylation of BVdU to its 5 monophosphate (BVdUMP) by the virus-coded thymidine kinase. In a cell-free system, this monophosphate inhibited the transport of pyrimidine sugar nucleotides across Golgi membranes and, as a consequence, the incorporation of sugars into glycoproteins. Inhibition of galactosyltransferase by BVdUMP was insignificant. BVdUMP did not inhibit translocation across the Golgi membrane of purine sugar nucleotides. Inhibition of sugar nucleotide translocation represents the first target for design of virus-specific glycosylation inhibitors.

S14.2 Topography of posttranslational modifications in yeast and mammals

In mammalian cells newly synthesized lysosomal proteins are targeted to lysosomes by two different mechanisms. Mannose-6-phosphate residues are transferred to soluble lysosomal hydrolases during their passage through the Golgicomplex. In the Trans-Golgi-Network specific receptors recognize these carbohydrate markers. The receptor ligand complexes are sorted into clathrin coated vesicles which are fused with elements of the endocytotic pathway. Due to the acidic pH within the endosomes the lysosomal hydrolases dissociate from the receptors and are transported to lysosomes via an as yet unknown mechanism. Processing of the polypeptide and carbohydrate chains of the lysosomal proteins takes place on their way to and within lysosomes. The receptors recycle to the Golgi for another round of transport or to the plasma membrane. At the cell surface the endocytosis of lysosomal enzymes can be mediated. In the internalization of the receptors from the plasma membrane cytosolic proteins are involved which recognize signals in the cytoplasmic domains of the receptors. They mediate the concentration of the receptors in clathrin-coated pits. Transport of membrane proteins depends on the signals that reside within their cytoplasmic tail. This is exemplified for the lysosomal acid phosphatase which is synthesized and transported as an integral membrane protein to lysosomes via the plasma membrane. The cytoplasmic proteins mediate the sorting at the TGN and the plasma membrane have distinct structural requirements for binding. Among the cytoplasmic receptors recognizing sorting signals within the cytoplasmic tails of lysosomal membrane glycoproteins and mannose-6phosphate receptors the HA1 and HA2 adaptors have been identified.