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: 4.4). 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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    • "The GRIP domain proteins can form α-helical parallel homodimers [26], in which each subunit interacts separately with one Arl1-GTP, and the Golgi localization of GRIP domain proteins in animal and yeast cells is dependent on the presence of Arl1 [17], [18]. GRIP domain dimerization is critical for Golgi targeting because the disruption of this dimerization's formation results in the loss of Golgi targeting. "
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    ABSTRACT: Background In animals and fungi, dimerization is crucial for targeting GRIP domain proteins to the Golgi apparatus. Only one gene in the Arabidopsis genome, AtGRIP, codes for a GRIP domain protein. It remains unclear whether AtGRIP has properties similar to those of GRIP domain proteins. Results In this study, western blot and yeast two-hybrid analyses indicated that AtGRIPs could form a parallel homodimer. In addition, yeast two-hybrid analysis indicated that AtGRIPaa711–753, AtGRIPaa711–766 and AtGRIPaa711–776 did not interact with themselves, but the intact GRIP domain with the AtGRIP C-terminus did. Confocal microscopy showed that only an intact GRIP domain with an AtGRIP C-terminus could localize to the Golgi stacks in Arabidopsis leaf protoplasts. A BLAST analysis showed that the C-terminus of GRIP proteins was conserved in the plant kingdom. Mutagenesis and yeast two-hybrid analyses showed that the L742 of AtGRIP contributed to dimerization and was crucial for Golgi localization. Conclusions These results indicate that the C-terminus of GRIP proteins is essential for self-association and for targeting of Golgi stacks in plant cells. We suggest that several properties of GRIP proteins differ between plant and animal cells.
    PLoS ONE 06/2014; 9(6):e98963. DOI:10.1371/journal.pone.0098963 · 3.23 Impact Factor
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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 · 8.68 Impact Factor
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    • "Constructs A construct containing the C-terminal 82 amino acids of GCC185 (residue 1603 to 1684) fused to GFP (GFP-GCC185 GRIP ) has been described (Luke et al., 2005). The sequence encoding the C-terminal 200 amino acids of GCC185 (residues 1485–1684) was amplified from pBluescript-SK + -KIAA0336 (provided by Kazusa DNA Research Institute, Kisarazu, Chiba, Japan) using the primers 5 0 -CCGGAATTCCGTTTCCTCTCAACAATCT-3 0 and 5 0 -CCGGAA TTCCTATCGAAGTCCAGACCA-3 0 . "
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    ABSTRACT: Mammalian golgins of the trans-Golgi network (TGN) are small G protein effectors that are required for membrane transport and contain a Golgi targeting C-terminal GRIP domain. The localization of two TGN golgins, p230/golgin-245 and golgin-97, is mediated by the small GTPase Arl1, whereas recruitment of the TGN golgin GCC185 is controversial. Recently, GCC185 was proposed to localize to the Golgi by the co-operation of two small GTPases, Rab6A/A' and Arl1 (Burguete et al., 2008), a model based predominantly on in vitro interactions. Here we demonstrate that Golgi recruitment of endogenous GCC185 does not involve Rab6A/A' and Arl1. We find minimal colocalization between Rab6A/A' and endogenous GCC185 on Golgi membranes and failed to detect an interaction between Rab6A/A' and C-terminal domains of GCC185 by yeast two-hybrid analyses. Moreover, depletion of both Rab6A/A' and Arl1 also had no effect on the localization of endogenous GCC185 or the isolated GRIP domain of GCC185.
    Cell 09/2009; 138(4):787-94. DOI:10.1016/j.cell.2009.05.048 · 32.24 Impact Factor
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