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Identification and characterization of Golgins

In recent years, a large number of coiled-coil proteins localised to the Golgi apparatus have been identified using antisera from human patients with a variety of autoimmune conditions. Because of their common method of discovery and extensive regions of coiled-coil, they have been classified as a family of proteins, the golgins. This family includes golgin-230/245/256, golgin-97, GM130/golgin-95, golgin-160/MEA-2/GCP170, giantin/macrogolgin and a related group of proteins - possibly splice variants - GCP372 and GCP364. GM130 and giantin have been shown to function in the p115-mediated docking of vesicles with Golgi cisternae. In this process, p115, another coiled-coil protein, is though to bind to giantin on vesicles and to GM130 on cisternae, thus acting as a tether holding the two together. Apart from giantin and GM130, none of the golgins has yet been assigned a function in the Golgi apparatus. In order to obtain clues as to the functions of the golgins, the targeting to the Golgi apparatus of two members of this family, golgin-230/245/256 and golgin-97, was investigated. Each of these proteins was shown to target to the Golgi apparatus through a carboxy-terminal domain containing a conserved tyrosine residue, which was critical for targeting. The domain preferentially bound to Rab6 on protein blots, and mutations that abolished Golgi targeting resulted in a loss of this interaction. Sequence analysis revealed that a family of coiled-coil proteins from mammals, worms and yeast contain this domain at their carboxyl termini. One of these proteins, yeast Imh1p, has previously been shown to have a tight genetic interaction with Rab6. On the basis of these data, it is proposed that this family of coiled-coil proteins functions in Rab6-regulated membrane-tethering events (Barr, 1999).

The mechanism by which peripheral membrane proteins are targeted to the cytoplasmic face of the Golgi apparatus is poorly understood. A carboxy-terminal domain of the trans-Golgi-network (TGN) protein, p230, has been identified that is responsible for Golgi localisation. This study reports the identification of a similar Golgi-localisation domain (GLD, also termed the 'GRIP' domain) in a family of putative peripheral membrane proteins from lower and higher eucaryotes. The majority of family members have a domain structure similar to that of p230, with extensive coiled-coil regions (>80%) and the potential GLD located in a non-coiled-coil domain at the carboxyl terminus. Previously reported proteins in this family include human golgin-97 and Saccharomyces cerevisiae Imh1p. By constructing chimeric cDNAs encoding carboxy-terminal regions of these family members fused to green fluorescent protein (GFP), it has been directly demonstrated that the GLD of p230, golgin-97, the newly identified human protein GCC1p and yeast Imh1p functions as a Golgi-targeting domain in transfected mammalian cells. Site-directed mutagenesis of the GLDs identified two conserved aromatic residues that are critical for the function of this targeting domain. Endogenous p230 was displaced from the Golgi membranes in transfected cells expressing high levels of GFP fused to the GLD of either p230 or golgin-97, indicating that different GLDs interact with similar membrane determinants. Thus, a family of coiled-coil proteins has been identified that share a domain shown to be sufficient for the localisation of peripheral membrane proteins to the Golgi apparatus (Kjer-Nielsen, 1999).

Structure of golgins

tGolgin-1 (golgin-245, trans golgi p230) and golgin-97 are members of a family of peripheral membrane proteins of unknown function that localize to the trans Golgi network (TGN) through a conserved C-terminal GRIP domain. GRIP protein function was probed for by assessing the consequences of overexpressing isolated GRIP domains. By semi-quantitative immunofluorescence microscopy it was found that high level expression of epitope-tagged, GRIP domain-containing fragments of tGolgin-1 or golgin-97 specifically altered the characteristic pericentriolar distribution of TGN integral membrane and coat components. Concomitantly, vesicular transport from the TGN to the plasma membrane and furin-dependent cleavage of substrate proteins in the TGN were inhibited. Mutagenesis of a conserved tyrosine in the tGolgin-1 GRIP domain abolished these effects. GRIP domain overexpression had little effect on the distribution of most Golgi stack resident proteins and no effect on markers of other organelles. Electron microscopy analyses of GRIP domain-overexpressing cells revealed distended perinuclear vacuoles and a proliferation of multivesicular late endosomes to which the TGN resident protein TGN46 was largely mislocalized. These studies, the first to address the function of GRIP domain-containing proteins in higher eukaryotes, suggest that some or all of these proteins and/or their ligands function in maintaining the integrity of the TGN by regulating resident protein localization (Yoshino, 2003).

A recently described family of TGN (trans-Golgi network) proteins, all of which contain a GRIP domain targeting sequence, has been proposed to play a role in membrane transport. On the basis of the high content of heptad repeats, GRIP domain proteins are predicted to contain extensive coiled-coil regions that have the potential to mediate protein-protein interactions. Four mammalian GRIP domain proteins have been identified which are targeted to the TGN through their GRIP domains, namely p230, golgin-97, GCC88 and GCC185. This study investigated the ability of the four mammalian GRIP domain proteins to interact. Using a combination of immunoprecipitation experiments of epitope-tagged GRIP domain proteins, cross-linking experiments and yeast two-hybrid interactions, it has been established that the GRIP proteins can self-associate to form homodimers exclusively. Two-hybrid analysis indicated that the N- and C-terminal fragments of GCC88 can interact with themselves but not with each other, suggesting that the GRIP domain proteins form parallel coiled-coil dimers. Analysis of purified recombinant golgin-97 by CD spectroscopy indicated a 67% alpha-helical structure, consistent with a high content of coiled-coil sequences. These results support a model for GRIP domain proteins as extended rod-like homodimeric molecules. The formation of homodimers, but not heterodimers, indicates that each of the four mammalian TGN golgins has the potential to function independently (Luke, 2005).

Golgin-97, RanBP2alpha, Imh1p and p230/golgin-245 (GRIP) domain golgins are targeted to the Golgi membrane through their GRIP domains. By analyzing more than 30 mutants of golgin-97 and golgin-245 GRIP domains for their properties of dimerization, interaction with ARF like protein 1 (Arl1)-GTP and Golgi targeting, hierarchically organized three-tier interactions were found governing the Golgi targeting of GRIP domain golgins. GRIP domain self-dimerization is necessary for bivalent interaction with Arl1-GTP. Unexpectedly, however, these two interactions are not sufficient for Golgi targeting, as a third group of residues, including positive-charged arginine between alpha1 and alpha2 and hydrophobic residues C-terminal to the GRIP domain, turn out to be essential. Surface plasmon resonance analysis indicates that GRIP domain interacts directly with membrane lipid, partially through the third group of residues such as W744 of golgin-97. This third tier of interaction with the membrane could be mediated by non-specific hydrophobic and electrostatic forces (Lu, 2006).

Arl1-GTP recruits Golgin-97 onto the Golgi

A cellular role and the mechanism of action for small GTPase Arl1 have been defined. Arl1-GTP interacts with the GRIP domains of Golgin-97 and Golgin-245, a process dependent on conserved residues of the GRIP domains that are important for Golgi targeting. The switch II region of Arl1 confers the specificity of this interaction. Arl1-GTP mediates Golgi recruitment of Golgin-97 in a switch II-dependent manner, whereas tethering Arl1-GTP onto endosomes can mediate endosomal targeting of Golgin-97. Golgin-97 and Golgin-245 are dissociated from the Golgi when Arl1 is knocked-down by its siRNA. Arl1-GTP thus functions to recruit Golgin-97 and Golgin-245 onto the Golgi via interacting with their GRIP domains (Lu, 2003).

Golgin-97 acts in endosome-to-TGN transport

The precise cellular function of Arl1 and its effectors, the GRIP domain Golgins, is not resolved, despite the recent understanding that Arl1 regulates the membrane recruitment of these Golgins. This report describes a functional study of Golgin-97. Using a Shiga toxin B fragment (STxB)-based in vitro transport assay, it has been demonstrated that Golgin-97 plays a role in transport from the endosome to the trans-Golgi network (TGN). The recombinant GRIP domain of Golgin-97 as well as antibodies against Golgin-97 inhibit the transport of STxB in vitro. Membrane-associated Golgin-97, but not its cytosolic pool, is required in the in vitro transport assay. The kinetic characterization of inhibition by anti-Golgin-97 antibody in comparison with anti-Syntaxin 16 antibody established that Golgin-97 acts before Syntaxin 16 in endosome-to-TGN transport. Knock down of Golgin-97 or Arl1 by their respective small interference RNAs (siRNAs) also significantly inhibits the transport of STxB to the Golgi in vivo. In siRNA-treated cells with reduced levels of Arl1, internalized STxB was instead distributed peripherally. Microinjection of Golgin-97 antibody led to the fragmentation of Golgi apparatus and the arrested transport to the Golgi of internalized Cholera toxin B fragment. This study suggests that Golgin-97 may function as a tethering molecule in endosome-to-TGN retrograde traffic (Lu, 2004).

Transport of E-cadherin requires golgin-97

E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. This study examined the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN (Lock, 2005).

The centrosome nucleates the Golgi apparatus through Golgin-97

The Golgi apparatus breaks down at mitosis, resulting in the dispersal of Golgi-resident proteins. In NRK cells, however, subsets of both TGN38 and golgin-97, but not ManII and GM130, remained associated with the centrosome throughout the cell cycle. This centrosome association of TGN38 and golgin-97 was not disrupted by treatment with brefeldin A, additional inducers of retrograde trafficking and inhibitors of either kinases or protein phosphatases. Anchoring of the Golgi apparatus within the juxtanuclear region depends on microtubules; the association of TGN38 and golgin-97 subsets with the centrosome, however, was insensitive to nocodazole treatment. Drugs such as PDMP, which block Golgi dispersal both by nocodazole, despite microtubule depolymerization, and by inducers of retrograde trafficking, strengthened the microtubule-nucleating activity of the centrosome. These observations cumulatively suggest the centrosome is implicated in nucleation of the Golgi apparatus through interactions with Golgi-resident proteins, such as TGN38 and golgin-97 (Takatsuki, 2002).

Golgin-97 is essential for virus replication

Poxviruses are the only DNA viruses known to replicate and assemble in the cytoplasm of infected cells. Poxvirus morphogenesis is a complicated process in which four distinct infectious forms of the virus are produced: intracellular mature virus, intracellular enveloped virus, cell-associated enveloped virus, and extracellular enveloped virus. The source of primary membrane wrapping the intracellular mature virus, the first infectious form, is still unknown. Although the membrane was suggested to originate from the endoplasmic reticulum-Golgi intermediate compartment, none of the marker proteins from this or any other cell compartments has been found in the intracellular mature virus. Thus, it was hypothesized that the membrane is either extensively modified by the virus or synthesized de novo. In the work described in this study, it has been demonstrated that a host cell protein residing in the trans-Golgi network membrane, golgin-97, is transported to the sites of virus replication and assembly and becomes incorporated into the virions during poxvirus infection. Inside the virion, golgin-97 is associated with the insoluble core protein fraction. Being able to adopt a long rod-like structure, the protein apparently extends through the virion envelope and protrudes from its surface. The potential role and functions of golgin-97 in poxvirus replication are discussed and working models are proposed (Alzhanova, 2006).

Acquisition of the membrane and genome encapsidation is an important step in the replication of enveloped viruses. The biogenesis of the poxviral primary membrane and the core as well as the mechanisms of their maturation are poorly understood. Using RNA interference approach, a cellular trans-Golgi network membrane protein, golgin-97, is essential for virus replication. Analysis of the virion morphology in the cells depleted of golgin-97 shows that the protein is required for the virus morphogenesis and, in particular, for the formation of the first infectious virus form, mature virus, but not its precursor, immature virus. This suggests that golgin-97 may be involved in the maturation of the virus core and, potentially, the virus membrane (Alzhanova, 2007).

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