TMF is a golgin that binds Rab6 and influences Golgi morphology

Abstract

Background: Golgins are coiled-coil proteins associated with the Golgi apparatus, that are believed to be involved in the tethering of vesicles and the stacking of cisternae, as well as other functions such as cytoskeletal association. Many are peripheral membrane proteins recruited by GTPases. Several have been described in animal cells, and some in yeast, but the relationships between golgins from different species can be hard to define because although they share structural features, their sequences are not well conserved.

Results: We show here that the yeast protein Sgm1, previously shown to be recruited to the Golgi by the GTPase Ypt6, binds to Ypt6:GTP via a conserved 100-residue coiled-coil motif that can be identified in a wide range of eukaryotes. The mammalian equivalent of Sgm1 is TMF/ARA160, a protein previously identified in various screens as a putative transcription or chromatin remodelling factor. We show that it is a Golgi protein, and that it binds to the three known isoforms of the Ypt6 homologue Rab6. Depletion of the protein by RNA interference in rat NRK cells results in a modest dispersal of Golgi membranes around the cell, suggesting a role for TMF in the movement or adherence of Golgi stacks.

Conclusion: We have identified TMF as an evolutionarily conserved golgin that binds Rab6 and contributes to Golgi organisation in animal cells.

Figure 1

Mapping the Ypt6 binding site on Sgm1 A. Coiled-coil probabilities of the Sgm1 protein sequence, calculated by the method of Lupas using the coils program. The regions analysed for binding are shown diagrammatically below. B. Immunoblot with anti-Protein A of the indicated portions of Sgm1 bound to and eluted from GST-Ypt6 in the GTP and GDP state. Input samples are yeast extract corresponding to about 1% of the total input. The intact protein A fusions are indicated by the arrowheads. Asterisks mark C terminal proteolytic fragments, still fused to protein A, that are common to both the full-length and 488–707 constructs; note that the smallest visible fragment corresponds closely to the 597–707 construct. Proteins apparently larger than the input in the 488–707 lanes presumably represent dimers and other aggregates.

Figure 2

Sgm1 homologues are present in a wide range of eukaryotes A. Coiled-coil predictions for various Sgm1-like proteins. Note that each has a distinct 100-residue coiled-coil domain at the C terminus, which in Sgm1 corresponds to the Ypt6-binding domain. The regions of TMF used for binding and antibody production are indicated by lines. The accession numbers for the uncharacterised proteins shown are: Neurospora, CAB97305; Arabidopsis, C96829; Drosophila AAF46211. B. Sequence alignment of the C-terminal coiled coil domains of the proteins depicted in A.

Figure 3

Binding of TMF to Rab6 isoforms A. Immunoblot showing the binding of the His-tagged C-terminal 310 residues of TMF to various GTPases, performed as in Figure 1. B. Coomassie stained gels showing the GTPases used in A, after elution from glutathione-Sepharose.

Figure 4

Location and function of TMF A. Immunoblot of Golgi and cytosol fractions with anti-TMF antibodies. The Golgi lane contained approximately 5 μg total protein, the cytosol lane approximately 100 μg protein. Marker positions (kDa) are indicated. B. Immunofluorescent images of COS cells showing endogenous TMF in an untransfected cell (untransf.), HA-tagged full-length protein expressed from a transfected plasmid (HA-TMF) and detected with both anti-HA and anti-TMF, as well as a cell expressing rather higher levels of HA-TMF (only the anti-HA image is shown; anti-TMF staining of this cell gave an identical pattern). The last panel shows the HA-tagged 310 C-terminal residues of HA (HA-C term) expressed similarly and detected with anti-HA. Bar is 10 μm. C. Rat NRK cells transfected with RNAi and stained for TMF and either TGN38 or GOS28 as indicated. Note that TMF-depleted cells (green in the merged images) have more dispersed Golgi than the other cells. The insets in some panels show an enlarged region of the Golgi, boxed in the merged image. Bars are 10 μm.