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The trans-Golgi network GRIP-domain proteins form alpha-helical homodimers. Biochem J 388: 835-841

The Russell Grimwade School of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia.
Biochemical Journal (Impact Factor: 4.78). 07/2005; 388(Pt 3):835-41. DOI: 10.1042/BJ20041810
Source: PubMed

ABSTRACT 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. In the present study, we have 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, we have 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.

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    • "Of the longer, coiled coil class of tethering proteins, there are at least five representatives anchored at the TGN: four GRIP domain tethers (Golgin 245, Golgin 97, GCC185, and GCC88) and TMF/ARA160 (Yamane et al. 2007). The former class use their carboxy-terminal GRIP domains as a binding site for the TGN localized, small GTPase, Arl1 (Panic et al. 2003; Munro 2005; Munro 2011); they are thought to form parallel homodimers that partition between the cytosol and the Golgi membrane surface (Luke et al. 2005). Golgin245 and Golgin 97 bind Arl1 tightly; GCC185 binds Arl1 with enhanced affinity in the presence of Rab6 GTPase (Burguete et al. 2008; Pfeffer 2009). "
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    ABSTRACT: The trans-Golgi network (TGN) receives a select set of proteins from the endocytic pathway-about 5% of total plasma membrane glycoproteins (Duncan and Kornfeld 1988). Proteins that are delivered include mannose 6-phosphate receptors (MPRs), TGN46, sortilin, and various toxins that hitchhike a ride backward through the secretory pathway to intoxicate cells after they exit into the cytoplasm from the endoplasmic reticulum (ER). This article will review work on the molecular players that drive protein transport from the endocytic pathway to the TGN. Distinct requirements have revealed multiple routes for retrograde transport; in addition, the existence of multiple, potential coat proteins and/or cargo adaptors imply that multiple vesicular transfers are likely involved. Several comprehensive reviews have appeared recently and should be sought for additional details (Bonifacino and Rojas 2006; Johannes and Popoff 2008).
    Cold Spring Harbor perspectives in biology 03/2011; 3(3). DOI:10.1101/cshperspect.a005272 · 8.23 Impact Factor
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    • "Consistent with this finding, chemical crosslinking of RBD-87 yielded efficient conversion to a dimer in solution (Figure 2D). Full-length GCC185 is also dimeric (Luke et al., 2005), consistent with the RBD-87 polypeptide forming a helical dimer. A helical wheel projection of the predicted coiled coil spanning the Rab-binding domain identified the hydrophobic face of an amphipathic helix, with residues at heptad positions 'a' and 'd' expected to reside at the interface of a dimeric coiled coil (Harbury et al., 1993; Figure 3A). "
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    ABSTRACT: 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.
    Cell 02/2008; 132(2):286-98. DOI:10.1016/j.cell.2007.11.048 · 33.12 Impact Factor
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    • "In contrast, the GRIP domains of GCC88 and GCC185, although dependent on G proteins for membrane binding, have different membrane binding properties from the GRIP domains of golgin-97 and p230/golgin-245 and they do not bind Arl1 in vivo (Derby et al., 2004), indicating different mechanisms of recruitment. Given that the four GRIP domain proteins form homodimers exclusively (Luke et al., 2005), each TGN golgin has the potential to function inde- This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07– 06 – 0622) on October 3, 2007. □ D The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). "
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    ABSTRACT: Retrograde transport pathways from early/recycling endosomes to the trans-Golgi network (TGN) are poorly defined. We have investigated the role of TGN golgins in retrograde trafficking. Of the four TGN golgins, p230/golgin-245, golgin-97, GCC185, and GCC88, we show that GCC88 defines a retrograde transport pathway from early endosomes to the TGN. Depletion of GCC88 in HeLa cells by interference RNA resulted in a block in plasma membrane-TGN recycling of two cargo proteins, TGN38 and a CD8 mannose-6-phosphate receptor cytoplasmic tail fusion protein. In GCC88-depleted cells, cargo recycling was blocked in the early endosome. Depletion of GCC88 dramatically altered the TGN localization of the t-SNARE syntaxin 6, a syntaxin required for endosome to TGN transport. Furthermore, the transport block in GCC88-depleted cells was rescued by syntaxin 6 overexpression. Internalized Shiga toxin was efficiently transported from endosomes to the Golgi of GCC88-depleted cells, indicating that Shiga toxin and TGN38 are internalized by distinct retrograde transport pathways. These findings have identified an essential role for GCC88 in the localization of TGN fusion machinery for transport from early endosomes to the TGN, and they have allowed the identification of a retrograde pathway which differentially selects TGN38 and mannose-6-phosphate receptor from Shiga toxin.
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