Monoclonal antibody hfd9 identifies a novel 28 kda integral membrane protein on the Cis-golgi

Abstract

We have raised a monoclonal antibody (mAb) (HFD9) that detects a 28 kDa protein (p28) enriched in the Golgi membrane. p28 was localized to the perinuclear Golgi region in all cell lines thus far examined. Its Golgi localization was confirmed by its colocalization with Golgi markers using indirect immunofluorescence microscopy. Immunogold labelling demonstrates that the majority of p28 was localized on the cis-Golgi and its associated structures. Two independent experiments demonstrate that the p28 epitope recognized by mAb HFD9 is exposed to the cytosol. Extraction of Golgi membranes with a variety of reagents revealed that p28 behaves like an integral membrane protein. mAb HFD9 thus defines a novel 28 kDa integral membrane protein on the cis-Golgi. To our knowledge, p28 represents the first integral membrane protein of the Golgi system identified via the antibody approach whose epitope is cytoplasmically-oriented and highly-conserved. Monoclonal antibody HFD9 will thus provide a useful tool for further studies on the cis side of the Golgi, which is not well characterised due to the lack of good markers.

Direct interaction between the COG complex and the SM protein, Sly1, is required for Golgi SNARE pairing

“…Antiserum against Cog8 was kindly provided by D Ungar (York University, York, UK), whereas polyclonal antibodies against GS28 and Syntaxin 5 and monoclonal anti‐VSV‐G antibody were a generous gift of Z Elazar (Weizmann Institute of Science, Rehovot, Israel). Polyclonal antibodies against GRASP‐65 (Peretti et al , 2008) and GS15 (Xu et al , 1997), and monclonal anti‐GS28 antibody (Subramaniam et al , 1995) were described earlier. Monoclonal anti‐GS15 and anti‐KDELR antibodies were purchased from BD Biosciences (San Jose, CA) and from Santa Cruz Biotechnology (Santa Cruz, CA), respectively.…”

Vti1a/b support distinct aspects of TGN and cis-/medial Golgi organization

“…It recruits Golgins to the Golgi and is required for a normal TGN structure 27 . Overexpression of dominant negative ARL1 leads to reduced levels of cis -Golgi protein GS28, but has no effect on GM130 30 – 32 . Since intensity of cis -/medial Golgi proteins is increased in Vti1a/b DKO neurons, loss of ARL1 cannot fully explain the Vti1a/b DKO phenotype.…”

A Model for the Self-Organization of Vesicular Flux and Protein Distributions in the Golgi Apparatus

“…2. The β SNARE Gos28 also decreases from cis to trans [26]; however, its cognate monomeric SNARE partner GS15 accumulates in the transmost cisternae instead [2], and the GS15 yeast homologue Sft1p is also enriched in the late Golgi [17], [23], (see Fig. 1C).…”