Golgins

They are designated golgin-84, -95, -160, -245, and -376 (giantin or macrogolgin) and were identified initially as human autoantigens (Chapter 31), appearing in the blood of persons with autoimmune disorders such as Sjogren s syndrome.281 282 Another protein... 

J., Satch, A., Pelletier, L., and Warren, G., Golgin tethers define subpopulations of COPI vesicles. Science 307,1095-1098,2005 Short, B Haas, A., and Barr, F.A., Golgins and GTPases, giving identity and structure to the Golgi apparatus, Biochim. Biophys. Acta 1744, 383-395, 2005 Satoh, A., Beard, M., and Warren, G., Preparation and characterization of recombinant golgin tethers. Methods Enzymol. 404, 279-296, 2005. 

Diao, A., Rahman, D., Pappin, D.J., Lucocq, J. and Lowe, M. (2003) The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation. J. Cell Biol. 160, 201-212. 

Caspase-2 lch-1 (human), Nedd2 (rat, mouse) Golgin-160, Lamins ( ) Apoptosis (activity suppressed by serum deprivation)... 

TETHERING ASSAYS FOR COPI VESICLES MEDIATED BY GOLGINS... 

A method is described that allows the attachment of COPI vesicles and Golgi membranes to glass slides that can then be analyzed using electron microscopy (EM) and immuno-EM methods. Subpopulations of COPI vesicles can be bound selectively using recombinant golgins. Alternatively, COPI vesicles can be attached to prebound Golgi membranes. Marking these vesicles selectively with biotin allows their site of attachment to be identified. 

In Vitro COPI Vesicle Tethering Assay Using Purified Golgins... 

In Vitro Tethering of COPI Vesicles to Golgin-Coated Glass Slides... 

Fig. 1. Tethering of COPI vesicles to CASP-coated slides. (A-E) The vesicle-containing supernatant was incubated for 30 min at 4° on glass slides precoated with (A, D, E) recombinant CASP, (B) 1% BSA and STI, or (C) recombinant EEAl in the absence (A, B, C) or presence of (D) soluble CASP or (E) soluble golgin-84. Glass slides were embedded in Epon resin and processed for conventional EM. (F) Input membranes. (G) Quantitation of the results in (A-E) presented as the mean of the number of the bound vesicles per fim S. D.(n=5). (Reprinted with permission from Science.)...

Note that uncoating the COPI vesicles is required for binding to golgin tethers (or Golgi membranes, see below) since binding was not observed when an ArflQVlL mutant was used for preparation of COPI vesicles (Malsam et al, 2005). 

Instead of biotinylation, fluorescently labeled COPI vesicles can be purified using this method. However, reagents such as NHS-Alexa-488 (Molecular Probes, A-10235) label many proteins in the COPI vesicles in addition to golgin-84. 

Barr, F. A, and Short, B. (2003). Golgins in the structure and dynamics of the Golgi apparatus. Curr. Opin. Cell Biol 15, 405-413. 

Bascom, R. A, Srinivasan, S., and Nussbaum, R. L. (1999). Identification and characterization of golgin-84, a novel Golgi integral membrane protein with a cytoplasmic coiled-coil domain. J. Biol Chem. 274, 2953-2962. 

Malsam, J., Satoh, A., Pelletier, L., and Warren, G. (2005). Golgin tethers define subpopulations of COPI vesicles. Science 307, 1095-1098. 

Golgins are minor components of Golgi membranes so that preparation of sufficient amounts for biochemical analysis is difficult. Here we describe the preparation of recombinant forms of those golgins that constitute the tethering complexes just described. 

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