The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation

Abstract

Fragmentation of the mammalian Golgi apparatus during mitosis requires the phosphorylation of a specific subset of Golgi-associated proteins. We have used a biochemical approach to characterize these proteins and report here the identification of golgin-84 as a novel mitotic target. Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks. Golgin-84 binds to active rab1 but not cis-Golgi matrix proteins. Overexpression or depletion of golgin-84 results in fragmentation of the Golgi ribbon. Strikingly, the Golgi ribbon is converted into mini-stacks constituting only ∼25% of the volume of a normal Golgi apparatus upon golgin-84 depletion. These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus. Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.

Regulation of Microtubule-dependent Recycling at theTrans-Golgi Network by Rab6A and Rab6A'

“…Similarly to TGN38, the trans-Golgi marker GalT lost its juxtanuclear Golgi staining before giantin ( Figure 2E). When comparing Mann-II or Golgin 84 with GM130, we saw a more rapid disappearance of the two more medial-markers (Rabouille et al, 1995;Diao et al, 2003;Satoh et al, 2003) than with the CGN/cis marker GM130 (Figure 2, G and H). It thus seems that Rab6 induces a Golgi-to-ER redistribution that occurs progressively with the trans-most cisternae disappearing first followed by the medial-cisternae and finally, the ciscisternae and the CGN, similar to previous observations made during nocodazole experiments (Yang and Storrie, 1998).…”

“…Primary antibodies used were as follows: rabbit polyclonal to rab6 C-19 peptide (Santa Cruz Biotechnology, Santa Cruz, CA); sheep polyclonal to human TGN46 (Serotec, Oxford, United Kingdom); mouse anti-TGN38 (Affinity Bioreagents, Golden, CO); mouse monoclonal against human transferrin receptor (Zymed Laboratories, South San Francisco, CA); mouse monoclonal ␥-adaptin (clone 100.3; Sigma-Aldrich); mouse monoclonal to early endosomal antigen-1 (EEA)-1 (BD Transduction Laboratories, Lexington, KY); lysosomal associated membrane protein-1 (LAMP1) (H4A3) monoclonal antibody (mAb) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA); mouse monoclonal 9E10 recognizing the myc tag epitope (Covance, Berkeley, CA) and mouse anti-␣-tubulin (DM1A; Sigma-Aldrich). Noncommercial antibodies used were rabbit anti-p24␥ 3 (gp27; Fullekrug et al, 1999), polyclonal anti-Sar1p antibody (Pepperkok laboratory; unpublished data), polyclonal anti-GFP (Karsenti laboratory; unpublished data) and from the following sources (given in parentheses): rabbit antisera to the cytoplasmic domain of human 46-kDa cation-dependent mannose 6-phosphate receptor (Annette Hille-Rehfeld, Gottingen University, Germany; Klumperman et al, 1993); mouse monoclonal 53FC3 against mannosidase II (Graham Warren, Yale University, New Haven, CT; Burke et al, 1983), rabbit anti-rat mannosidase II (Kelly Moremen, Georgia University, Athens, GA; Moremen and Touster, 1985), monoclonal anti-GalT (Ulla Mandel, Copenhagen University, Copenhagen, Denmark; Almeida et al, 1999), sheep anti-GM130 (Francis Barr, MPI-Martinsried, Germany; published data), sheep antigolgin-84 (Martin Lowe, Manchester University, Manchester, United Kingdom; Diao et al, 2003), rabbit anti-sec61␤ (Bernard Dobberstein, Heidelberg University, Heidelberg, Germany; High et al, 1993), monoclonal 5B10 anti-rab6 (Angelika Barnekow, Muenster University, Muenster, Germany; Schiedel et al, 1995), monoclonal 50.1 anti-dynamitin (Richard Vallee, Columbia University, New York, NY; Paschal et al, 1993), and rabbit polyclonal anti-KAP3 (Tobias Stauber, European Molecular Biology Laboratory, Heidelberg, Germany; unpublished data). Secondary antibodies to sheep, rabbit, and mouse conjugated to were obtained from Molecular Probes (Eugene, OR).…”

The Cargo Receptors Surf4, Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC)-53, and p25 Are Required to Maintain the Architecture of ERGIC and Golgi

“…Thus, this phenotype is different. Yet another type of Golgi fragmentation results from silencing golgin-84 (Diao et al, 2003). However, this phenotype is accompanied by changes of the ER, and it has been attributed to a defect in anterograde trafficking.…”

How the Golgi works: A cisternal progenitor model

“…The Golgi is decorated with so-called Golgin proteins that stabilize the ribbon and tether transport vesicles that dock there. Simple siRNA depletion of any one of these proteins leads to the formation of ministacks (14)(15)(16)(17)(18)(19). "GRIP" domain Golgins localize to the exit face of the Golgi, are predicted to be extended, coiled-coil structures, and they each contain multiple binding sites for Golgi Rab GTPases across their entire lengths (20,21).…”