GRIP domain-mediated targeting of two new coiled-coil proteins,GCC88 and GCC185, to subcompartments of the trans-Golgi network

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the trans-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of HeLa cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the trans-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.     

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno-electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230_GRIP fusion protein, we show binding specifically to a subset of membranes of the trans -Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230_GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230_GRIP-transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230_GRIP binds to a specific population of vesicles distinct from those labelled for β-COP or γ-adaptin. The GFP-p230_GRIP fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans -Golgi network tubulo-vesicular carriers.     

Membrane traffic: Arl GTPases get a GRIP on the Golgi

A subset of the golgin family of large coiled-coil proteins have a GRIP domain that mediates their localization to the trans-Golgi. Two recent papers show that the Arl3p and Arl1p small GTPases act sequentially to recruit GRIP domain proteins to the Golgi.     

The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi

Rud3p is a coiled-coil protein of the yeast cis-Golgi. We find that Rud3p is localized to the Golgi via a COOH-terminal domain that is distantly related to the GRIP domain that recruits several coiled-coil proteins to the trans-Golgi by binding the small Arf-like GTPase Arl1p. In contrast, Rud3p binds to the GTPase Arf1p via this COOH-terminal "GRIP-related Arf-binding" (GRAB) domain. Deletion of RUD3 is lethal in the absence of the Golgi GTPase Ypt6p, and a screen of other mutants showing a similar genetic interaction revealed that Golgi targeting of Rud3p also requires Erv14p, a cargo receptor that cycles between the endoplasmic reticulum and Golgi. The one human protein with a GRAB domain, GMAP-210 (CEV14/Trip11/Trip230), is known to be on the cis-Golgi, but the COOH-terminal region that contains the GRAB domain has been reported to bind to centrosomes and gamma-tubulin (Rios, R.M, A. Sanchis, A.M. Tassin, C. Fedriani, and M. Bornens. 2004. Cell. 118:323-335). In contrast, we find that this region binds to the Golgi in a GRAB domain-dependent manner, suggesting that GMAP-210 may not link the Golgi to gamma-tubulin and centrosomes.     

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by 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. Here, we examine 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.     

Golgins and GTPases, giving identity and structure to the Golgi apparatus

In this review we will focus on the recent advances in how coiled-coil proteins of the golgin family give identity and structure to the Golgi apparatus in animal cells. A number of recent studies reveal a common theme for the targeting of golgins containing the ARL-binding GRIP domain, and the related ARF-binding GRAB domain. Similarly, other golgins such as the vesicle tethering factor p115 and Bicaudal-D are targeted by the Rab GTPases, Rab1 and Rab6, respectively. Together golgins and their regulatory GTPases form a complex network, commonly known as the Golgi matrix, which organizes Golgi membranes and regulates membrane trafficking.     

A novel Golgi-localisation domain shared by a class of coiled-coil peripheral membrane proteins

The mechanism by which peripheral membrane proteins are targeted to the cytoplasmic face of the Golgi apparatus is poorly understood. Previously, we have identified a carboxy-terminal domain of the trans-Golgi-network (TGN) protein p230 that is responsible for Golgi localisation [1]. Here, we report the identification of a similar Golgi-localisation domain (GLD, also termed the 'GRIP' domain - see the paper by Munro and Nichols elsewhere in this issue) 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), we have 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, we have identified a family of coiled-coil proteins that share a domain shown to be sufficient for the localisation of peripheral membrane proteins to the Golgi apparatus.     

An Arabidopsis GRIP domain protein locates to the trans-Golgi and binds the small GTPase ARL1

GRIP domain proteins are a class of golgins that have been described in yeast and animals. They locate to the trans-Golgi network and are thought to play a role in endosome-to-Golgi trafficking. The Arabidopsis GRIP domain protein, AtGRIP, fused to the green fluorescent protein (GFP), locates to Golgi stacks but does not exactly co-locate with the Golgi marker sialyl transferase (ST)-mRFP, nor with the t-SNAREs Memb11, SYP31 and BS14a. We conclude that the location of AtGRIP is further to the trans side of the stack than STtmd-mRFP. The 185-aa C-terminus of AtGRIP containing the GRIP domain targeted GFP to the Golgi, although a proportion of the fusion protein was still found in the cytosol. Mutation of a conserved tyrosine (Y717) to alanine in the GRIP domain disrupted Golgi localization. ARL1 is a small GTPase required for Golgi targeting of GRIP domain proteins in other systems. An Arabidopsis ARL1 homologue was isolated and shown to target to Golgi stacks. The GDP-restricted mutant of ARL1, AtARL1-T31N, was observed to locate partially to the cytosol, whereas the GTP-restricted mutant AtARL1-Q71L labelled the Golgi and a population of small structures. Increasing the levels of AtARL1 in epidermal cells increased the proportion of GRIP-GFP fusion protein on Golgi stacks. We show, moreover, that AtARL1 interacted with the GRIP domain in a GTP-dependent manner in vitro in affinity chromatography and in the yeast two-hybrid system. This indicates that AtGRIP and AtARL1 interact directly. We conclude that the pathway involving ARL1 and GRIP domain golgins is conserved in plants.     

The GRIP domain - a novel Golgi-targeting domain found in several coiled-coil proteins

Many large coiled-coil proteins are being found associated peripherally with the cytoplasmic face of the organelles of the secretory pathway. Various roles have been proposed for these proteins, including the docking of donor vesicles or organelles to an acceptor organelle prior to fusion, and, in the case of the Golgi apparatus, the stacking of the cisternae [1] [2] [3] [4] [5]. Such critical roles require accurate recruitment to the correct organelle. For the endosomal coiled-coil protein EEA1, targeting requires a carboxy-terminal FYVE domain, which interacts with Rab5 and phosphatidylinositol 3-phosphate (PI(3)P), whereas the Golgi protein GM130 interacts with Golgi membranes via the protein GRASP65 [3] [6] [7]. In this paper, we show that two other mammalian Golgi coiled-coil proteins, golgin-245/p230 and golgin-97, have a conserved domain of about 50 amino acids at their carboxyl termini. This 'GRIP' domain is also found at the carboxyl terminus of several other large coiled-coiled proteins of unknown function, including two human proteins and proteins in the genomes of Caenorhabditis elegans and yeasts. The GRIP domains from several of these proteins, including that from the yeast protein Imh1p, were sufficient to specify Golgi targeting in mammalian cells when fused to green fluorescent protein (GFP). This result suggests that this small domain functions to recruit specific coiled-coil proteins to the Golgi by recognising a determinant that has been well conserved in eukaryotic evolution.     

Rab and Arl GTPase family members cooperate in the localization of the golgin GCC185

GCC185 is a large coiled-coil protein at the trans Golgi network that is required for receipt of transport vesicles inbound from late endosomes and for anchoring noncentrosomal microtubules that emanate from the Golgi. Here, we demonstrate that recruitment of GCC185 to the Golgi is mediated by two Golgi-localized small GTPases of the Rab and Arl families. GCC185 binds Rab6, and mutation of residues needed for Rab binding abolishes Golgi localization. The crystal structure of Rab6 bound to the GCC185 Rab-binding domain reveals that Rab6 recognizes a two-fold symmetric surface on a coiled coil immediately adjacent to a C-terminal GRIP domain. Unexpectedly, Rab6 binding promotes association of Arl1 with the GRIP domain. We present a structure-derived model for dual GTPase membrane attachment that highlights the potential ability of Rab GTPases to reach binding partners at a significant distance from the membrane via their unstructured and membrane-anchored, hypervariable domains.     

Golgi coiled-coil proteins contain multiple binding sites for Rab family G proteins

Vesicles and other carriers destined for the Golgi apparatus must be guided to the correct cisternae. Golgins, long coiled-coil proteins that localize to particular Golgi subdomains via their C termini, are candidate regulators of vesicle sorting. In this study, we report that the GRIP domain golgins, whose C termini bind the Arf-like 1 G protein on the trans-Golgi, can also bind four members of the Rab family of G proteins. The Rab2-, Rab6-, Rab19-, and Rab30-binding sites are within the coiled-coil regions that are not required for Golgi targeting. Binding sites for two of these Rabs are also present on two coiled-coil proteins of the cis-Golgi, the Drosophila melanogaster orthologues of GM130 and GMAP-210. We suggest an integrated model for a tentacular Golgi in which coiled-coil proteins surround the Golgi to capture and retain Rab-containing membranes, excluding other structures such as ribosomes. Binding sites for diverse Rabs could ensure that incoming carriers are captured on first contact and moved to their correct destination within the stack.     

Interaction between p230 and MACF1 is associated with transport of a glycosyl phosphatidyl inositol-anchored protein from the Golgi to the cell periphery

The molecular basis by which proteins are transported along cytoskeletal tracts from the trans-Golgi network (TGN) to the cell periphery remains poorly understood. Previously, using human autoimmune sera, we identified and characterized a TGN protein, p230/Golgin-245, an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, that is anchored to TGN membranes and TGN-derived vesicles by its carboxyl-terminal GRIP domain. To identify molecules that interact with the flexible amino-terminal end of p230, we used this domain as bait to screen a human brain cDNA library in a yeast two-hybrid assay. We found that this domain interacts with the carboxyl-terminal domain of MACF1, a protein that cross-links microtubules to the actin cytoskeleton. The interaction was confirmed by co-immunoprecipitation, an in vitro binding assay, double immunofluorescence images demonstrating overlapped localization in HeLa cells, and co-localization of FLAG-tagged constructs containing the interacting domains of these two proteins with their endogenous partners. Expression in HeLa cells of FLAG-tagged constructs containing the interacting domains of p230 and MACF1 disrupted transport of the glycosyl phosphatidyl inositol-anchored marker protein conjugated with yellow fluorescent protein (YFP-SP-GPI), while trafficking of the transmembrane marker protein, vesicular stomatitis virus glycoprotein conjugated with YFP (VSVG3-GL-YFP), was unaffected. Our results suggest that p230, through its interaction with MACF1, provides the molecular link for transport of GPI-anchored proteins along the microtubule and actin cytoskeleton from the TGN to the cell periphery.     

Arfaptins are localized to the trans-Golgi by interaction with Arl1, but not Arfs

Arfaptins (arfaptin-1 and arfaptin-2/POR1) were originally identified as binding partners of the Arf small GTPases. Both proteins contain a BAR (Bin/Amphiphysin/Rvs) domain, which participates in membrane deformation. Here we show that arfaptins associate with trans-Golgi membranes. Unexpectedly, Arl1 (Arf-like 1), but not Arfs, determines the trans-Golgi association of arfaptins. We also demonstrate that arfaptins interact with Arl1 through their BAR domain-containing region and compete for Arl1 binding with golgin-97 and golgin-245/p230, both of which also bind to Arl1 through their GRIP (golgin-97/RanBP2/Imh1p/p230) domains. However, arfaptins and these golgins show only limited colocalization at the trans-Golgi. Time-lapse imaging of cells overexpressing fluorescent protein-tagged arfaptins and golgin-97 reveals that arfaptins, but not golgin-97, are included in vesicular and tubular structures emanating from the Golgi region. These observations indicate that arfaptins are recruited onto trans-Golgi membranes by interacting with Arl1, and capable of inducing membrane deformation via their BAR domains.     

Golgins in the structure and dynamics of the Golgi apparatus

Golgins are a family of coiled-coil proteins associated with the Golgi apparatus necessary for tethering events in membrane fusion and as structural supports for Golgi cisternae. Recent work has shown that golgins such as GM130, golgin-45 and p115 bind to Rab GTPases via their coiled-coil domains, and that GM130, rather than being part of a static structural matrix, is in dynamic exchange between the membrane surface and the cytoplasm. Golgins such as bicaudal-D1 and -D2 bind to Rab6, but, rather than tethering membranes together, link vesicles to the cytoskeleton, thus adding a new function for this class of proteins. Other golgins containing the Golgi targeting GRIP domain, rather than binding Rabs, interact with and are recruited to membranes by another class of GTPase, the Arls. Current evidence therefore suggests that golgins function in a variety of membrane-membrane and membrane-cytoskeleton tethering events at the Golgi apparatus, and that all these are regulated by small GTPases of the Rab and Arl families.     

The 17-residue transmembrane domain of beta-galactoside alpha 2,6- sialyltransferase is sufficient for Golgi retention

beta-Galactoside alpha 2,6-sialyltransferase (ST) is a type II integral membrane protein of the Golgi apparatus involved in the sialylation of N-linked glycans. A series of experiments has shown that the 17-residue transmembrane domain of ST is sufficient to confer localization to the Golgi apparatus when transferred to the corresponding region of a cell surface type II integral membrane protein. Lectin affinity chromatography of chimeric proteins bearing this 17-residue sequence suggests that these chimeric proteins are localized in the trans-Golgi cisternae and/or trans-Golgi network. Further experiments suggest that this 17-residue sequence functions as a retention signal for the Golgi apparatus.     

The trans-Golgi network golgin, GCC185, is required for endosome-to-Golgi transport and maintenance of Golgi structure

Four mammalian golgins are specifically targeted to the trans-Golgi network (TGN) membranes via their C-terminal GRIP domains. The TGN golgins, p230/golgin-245 and golgin-97, are recruited via the GTPase Arl1, whereas the TGN golgin GCC185 is recruited independently of Arl1. Here we show that GCC185 is localized to a region of the TGN distinct from Arl1 and plays an essential role in maintaining the organization of the Golgi apparatus. Using both small interfering RNA (siRNA) and microRNA (miRNA), we show that depletion of GCC185 in HeLa cells frequently resulted in fragmentation of the Golgi apparatus. Golgi apparatus fragments were dispersed throughout the cytoplasm and contained both cis and trans markers. Trafficking of anterograde and retrograde cargo was analysed over an extended period following GCC185 depletion. Early effects of GCC185 depletion included a perturbation in the distribution of the mannose-6-phosphate receptor and a block in shiga toxin trafficking to the Golgi apparatus, which occurred in parallel with the fragmentation of the Golgi ribbon. Internalized shiga toxin accumulated in Rab11-positive endosomes, indicating GCC185 is essential for transport between the recycling endosome and the TGN. In contrast, the plasma membrane-TGN recycling protein TGN38 was efficiently transported into GCC185-depleted Golgi apparatus fragments throughout a 96-h period, and anterograde transport of E-cadherin was functional until a late stage of GCC185 depletion. This study demonstrated (i) a more effective long-term depletion of GCC185 using miRNA than siRNA and (ii) a dual role for the GCC185 golgin in the regulation of endosome-to-TGN membrane transport and in the organization of the Golgi apparatus.     

Multilayer interactions determine the Golgi localization of GRIP golgins

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, we found hierarchically organized three-tier interactions 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.     

Targeting of the Arf-like GTPase Arl3p to the Golgi requires N-terminal acetylation and the membrane protein Sys1p

The GTPase Arl3p is required to recruit a second GTPase, Arl1p, to the Golgi in Saccharomyces cerevisiae. Arl1p binds to the GRIP domain, which is present in a number of long coiled-coil proteins or 'golgins'. Here we show that Arl3p is not myristoylated like most members of the Arf family, but is instead amino-terminally acetylated by the NatC complex. Targeting of Arl3p also requires a Golgi membrane protein Sys1p. The human homologues of Arl3p (Arf-related protein 1 (ARFRP1)) and Sys1p (hSys1) can be isolated in a complex after chemical cross-linking. This suggests that the targeting of ARFRP1/Arl3p to the Golgi is mediated by a direct interaction between its acetylated N terminus and Sys1p/hSys1.     

A novel Rab6-interacting domain defines a family of Golgi-targeted coiled-coil proteins

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 [1]. Because of their common method of discovery and extensive regions of coiled-coil, they have been classified as a family of proteins, the golgins [1]. 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[2][3][4][5][6][7][8][9][10][11]. GM130 and giantin have been shown to function in the p115-mediated docking of vesicles with Golgi cisternae [12]. 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 [12] [13]. 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 [14]. On the basis of these data, it is proposed that this family of coiled-coil proteins functions in Rab6-regulated membrane-tethering events.     

Maintenance of the diacylglycerol level in the Golgi apparatus by the Nir2 protein is critical for Golgi secretory function

The level of diacylglycerol (DAG) in the Golgi apparatus is crucial for protein transport to the plasma membrane. Studies in budding yeast indicate that Sec14p, a phosphatidylinositol (PI)-transfer protein, is involved in regulating DAG homeostasis in the Golgi complex. Here, we show that Nir2, a peripheral Golgi protein containing a PI-transfer domain, is essential for maintaining the structural and functional integrity of the Golgi apparatus in mammalian cells. Depletion of Nir2 by RNAi leads to substantial inhibition of protein transport from the trans-Golgi network to the plasma membrane, and causes a reduction in the DAG level in the Golgi apparatus. Remarkably, inactivation of cytidine [corrected] 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis restores both effects. These results indicate that Nir2 is involved in maintaining a critical DAG pool in the Golgi apparatus by regulating its consumption via the CDP-choline pathway, demonstrating the interface between secretion from the Golgi and lipid homeostasis.