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.

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Abstract. Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme,/31,4 gaiactosyltransferase (GaiT) and the medial enzyme, N-acetylglucosaminylransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myctagged version of the endogenous protein. GAIT was found in the trans-cisterna and trans-Golgi network but, contrary to expectation, NAGT I was found both in the medial-and trans-cisternae, overlapping the distribution of GAIT. About one third of the NAGT I and half of the GaIT were found in the shared, transcisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.T HE construction of N-linked, bi-antennary oligosaccharides in the Golgi apparatus is a three-stage process.The first stage, thought to take place in the cis-Golgi network (CGN)l/cis-cisternae, continues the trimming of mannose residues started in the ER. The second stage, in medial-cisternae, involves addition of N-acetylglucosamine, the removal of a further two mannose residues and the addition of another N-acetylglucosamine. Fucose may also be added at this stage. The third and last stage, in the transcisternae and trans-Golgi network (TGN), involves addition of galactose followed by sialic acid (for review see Kornfeld and Kornfeld, 1985).These activities have been assigned to the different cisternae and networks based on localiTation of the appropriate enzymes and their products (for reviews see Rothman, 1985, andRoth, 1987). The first stage enzyme, mannosidase I, is thought to reside in the CGN/cis-cisternae because it acts on oligosaccharides attached to proteins that have just left the ER (Balch et al., 1986) and before the second stage enzyme, N-acetylglucosaminyltransferase I (NAGT I), which has been immunolocalized to medialcisternae . Other second stage enzymes (mannosidase II and NAGT H) cofractionate with NAGT I on sucrose gradients and, since they can be partially separated from third-stage enzymes (/31,4 galactosyltransferase [GAIT] and ot2,6 sialyltransferase [SialylT]) on the same gradients (Dunphy et al., 1981; Dunphy and Rothman, Tommy Nilsson and Marc Pypaert contributed equally to this work.

Abstract. Rat liver Golgi stacks were extracted with

The surprtsing result that the spanning domain causes retention of proteins in the Golgl stack poses the question as to the actual mechanism.Here we present a simple model that might have general applicability.