Characterization of a cis-Gol Matrix Protein , GM 130

Antisera raised to a detergentand 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 medialand 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. T HE Golgi apparatus receives the entire output of newly-synthesized proteins from the ER. They enter at the cis or entry face, in a compartment termed the cis-Golgi network (CGN) ~ which can be thought of as the last quality control step on the pathway (Huttner and Tooze, 1989; Pelham, 1991). Only properly folded proteins are allowed to proceed on to the stacked cisternae (Hurtley and Helenius, 1989) where posttranslational modifications are carried out, most notably to the bound oligosaccharides (Roth, 1987). When they reach the TGN, the proteins are sorted to their correct destination (Griffiths and Simons, 1986). Considerable progress has been made in identifying the proteins involved in the vesicle machinery responsible for conveying cargo from one compartment to the next (Pryer Address all correspondence to G. Warren, Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A, 3PX, UK. (Ph.): (44) 171 269 3561. Fax: (44) 171 269 3417. e.mail: warren@europa.lif. icnet.uk. T. Nilsson's present address is European Molecular Biology Laboratory, 69012 Heidelberg, Germany. P. Slusarewicz's present address is Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3844. 1. Abbreviations used in this paper: BFA, Brefeldin A; CGN, cis-Golgi network; immuno-EM; immunoelectron microscopy; Man II, al,3-1,6mannosidase II; NAGT I, 13-1,2-N-acetylglucosaminyltransferase I; NRK cells, normal rat kidney cells; RACE, Rapid Amplification of cDNA End; SialylT, u2,6-sialyltransferase; TGN, trans-Golgi network; VSV-G, vesicular stomatitis virus glycoprotein. et al., 1992; Rothman, 1994). The same is also true for those enzymes that carry out the posttranslational modifications. Many have now been sequenced and the signals that determine their precise location within the Golgi apparatus are being investigated (see Nilsson and Warren, 1994). Much less progress has been made in identifying structural proteins responsible for the unique architecture of the Golgi apparatus. Morphological studies have shown that there are structures in the space between the cisternae that might be involved in cisternal stacking (Franke et al., 1972; Mollenhauer, 1965; Mollenhauer et al., 1973; Cluett and Brown, 1992). Each Golgi apparatus is also embedded in a "zone of exclusion" containing many fibriUar structures (Mollenhauer and Morr6, 1978). It is not, however, clear to what extent these structures can be matched to the increasing number of cytoplasmic proteins that are now known to be associated with the Golgi apparatus. These proteins are of several types. There are those that interact with the classical cytoskeleton or are homologues of it. Comitin, an aetin-binding protein, has been localized to the periphery of the Golgi apparatus and may be involved in tethering the organelle to the actin cytoskeleton (Weiner et al., 1993). A homologue of the erythrocyte [3-spectrin has been localized to the Golgi apparatus (Beck et al., 1994b) as well as a homologue of ankyrin (Beck et al., 1994a). Given the role of the parental proteins in maintaining the structure of the erythrocyte, these homologues might be involved in maintaining the structural integrity of the Golgi apparatus. © The Rockefeller University Press, 0021-9525/95/12f 1715/12 $2.00 The Journal of Cell Biology, Volume 131, Number 6, Part 2, December 1995 1715-1726 1715 on A uust 3, 2017 jcb.rress. org D ow nladed fom The second type of protein belongs to a group of human autoantigens. Most are autoantigens found in Sjrgren's syndrome, and comprise a 230-kD protein found on the trans side of the Golgi apparatus (Kooy et al., 1992), a 210kD protein found on the cis side (Rios et al., 1994) and two other proteins (golgin-95 and golgin-160) which are associated with the Golgi apparatus though the precise location is still unclear (Fritzler et al., 1993). The sequences of both golgin-95 and golgin-160 have been determined and reveal a high content of heptad repeats suggesting a rodlike structure (Fritzler et al., 1993). Giantin/GCP372 is also an autoantigen found in rheumatoid arthritis. It has a high content of heptad repeats and a molecular weight in excess of 350 kD (Linstedt and Hauri, 1993; Seelig et al., 1994; Sohda et al., 1994). Unlike the other autoantigens found so far it is a membrane protein that is anchored by a COOH-terminal spanning domain. It has been suggested that this protein might be involved in cisternal stacking, but immunoelectron microscopy (immuno-EM) indicates that most of the protein surrounds the Golgi stacks (Seelig et al., 1994). It is, therefore, more likely to act as a tethering or positioning device. Our approach to identifying structural proteins of the Golgi has been to prepare a detergentand salt-resistant matrix from highly purified rat liver Golgi stacks (Slusarewicz et al., 1994b). One property of this matrix is the capacity to bind medial-/trans-Golgi enzymes with high affinity. Since these enzymes are present throughout individual Golgi cisternae and the binding is sensitive to prior treatment with proteases, this suggests that at least part of this matrix is present within the intercisternal space. Here we have used an antiserum to this matrix to screen an expression library and describe the properties of a 130kD protein (termed GM130 for Golgi matrix protein of 130 kD) which is related to golgin-95 and may be a new family member. Materials and Methods