Abstract. Antisera raised to a detergent-and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial-and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER-Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure.

A cell-free system that mimics the reassembly of Golgi stacks at the end of mitosis requires two ATPases, NSF and p97, to rebuild Golgi cisternae. Morphological studies now show that alpha-SNAP, a component of the NSF pathway, can inhibit the p97 pathway, whereas p47, a component of the p97 pathway, can inhibit the NSF pathway. Anti-syntaxin 5 antibodies and a soluble, recombinant syntaxin 5 inhibited both pathways, suggesting that this t-SNARE is a common component. Biochemical studies confirmed this, showing that p47 binds directly to syntaxin 5 and competes for binding with alpha-SNAP. p47 also mediates the binding of p97 to syntaxin 5 and so plays an analogous role to alpha-SNAP, which mediates the binding of NSF.

The early Golgi t-SNARE (target-membrane-associated soluble-N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 5 is thought to specify the docking site for both COPI and COPII coated vesicles originating from the endoplasmic reticulum (ER) and COPI vesicles on the retrograde pathway. We now show that there are two forms of syntaxin 5 that appear to be generated from the same mRNA by alternative initiation of translation. The short form (35 kDa) corresponds to the published sequence. The long form (42 kDa) has an N-terminal cytoplasmic extension containing a predicted type II ER retrieval signal. When grafted onto a reporter molecule, this signal localized the construct to the ER. Biochemical fractionation and immunofluorescence microscopy showed that there was less of the long form in the Golgi apparatus and more in peripheral punctate structures, some of which colocalized with markers of the intermediate compartment. The predicted absence of the long form in budding yeast points to a function unique to higher organisms.

Abstract. Rat liver Golgi stacks were extracted with

The distribution of beta 1,2 N-acetylglucosaminyltransferase I (NAGT I), alpha 1,3-1,6 mannosidase II (Mann II), beta 1,4 galactosyltransferase (GalT), alpha 2,6 sialyltransferase (SialylT) was determined by immuno-labelling of cryo-sections from HeLa cell lines. Antibody labelling in the HeLa cell line was made possible by stable expression of epitope-tagged forms of these proteins or forms from species to which specific antibodies were available. NAGT I and Mann II had the same distribution occupying the medial and trans cisternae of the stack. GalT and SialylT also had the same distribution but they occupied the trans cisterna and the trans-Golgi network (TGN). These results generalise our earlier observations on the overlapping distribution of Golgi enzymes and show that each of the trans compartments of the Golgi apparatus in HeLa cells contains unique mixtures of those Golgi enzymes involved in the construction of complex, N-linked oligosaccharides.

Using a series of chimeric and truncated N-acetylglucosaminyltransferase I (NAGT I) molecules we have shown that part of the lumenal stalk region is both necessary and sufficient for kin recognition of mannosidase II and retention in the Golgi stack. The membrane-spanning domain was not required for retention, but replacing part or all of this domain with leucine residues did have a dramatic effect on Golgi morphology. In stable cell lines, stacked cisternae were replaced by tubulo-vesicular clusters containing the mutated NAGT I. The loss of stacked cisternae was proportional to the number of leucines used to replace the membrane-spanning domain.

The use of peripheral blood (PB) progenitor cells to reconstitute hematopoiesis after high-dose chemotherapy is now common practice in the treatment of malignancies. Growth factors such as granulocyte colony-stimulating factor (G-CSF) induce high proliferation and self-renewal within the stem cell compartment of the bone marrow. This is followed by the release of a very high number of progenitor cells into the peripheral blood, the mechanism of which remains unclear. Previously, we have showed that high cell-surface sialylation reduces matured myeloid attachment to the bone marrow stroma, which is likely to help their release into the peripheral blood. Consequently, we decided to investigate the possibility that sialylation may also contribute to the CD34(+) cell release into the peripheral blood during G-CSF treatment. We found that G-CSF-mobilized PB CD34(+) cells exhibit an increase in the sialyltransferase ST6Gal-I mRNA level. The analysis of the heterogeneity of ST6Gal-I transcripts showed a predominance of the Y form in both bone marrow (BM) and G-CSF PB CD34(+) cells. Higher levels in ST6Gal-I mRNA resulted in a higher expression of alpha-2,6-sialylation on the surface of G-CSF PB CD34(+) cells when compared to noninduced PB CD34(+) cells but not higher to BM CD34(+) cells. Our present results, together with recent observations, suggest that ST6Gal-I and alpha-2,6-sialylation expression in myeloid cells is tightly dependent on the presence of G-CSF in their environment. Up-regulation of ST6Gal-I together with alpha-2,6-sialylation of cell surface glycoproteins is likely to modulate cellular adherence and survival of progenitor cells.

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