Immunoisolation of a Golgi subcompartment from Saccharomyces cerevisiae

• Main Author: Bryant, Nia J.
• Published: University of Edinburgh 1993
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.642161

Although no structure morphologically similar to the well-characterised Golgi complex of mammalian cells can be seen in <i>Saccharomyces cerevisiae</i> (yeast), there is much genetic and biochemical evidence to suggest the existence of a yeast organelle functionally equivalent to the mammalian Golgi. The <i>trans</i> Golgi network (TGN) of mammalian cells is characterised, at least in part, by the presence of serine proteases that process prohormone molecules by catalysing cleavage of polypeptide chains at pairs of basic residues. Analogous proprotein processing in yeast is achieved by the action of the Kex2 protease. Kex2 is the best-characterised resident protein of the yeast Golgi to date, and is believed to reside in a compartment of the organelle that is equivalent to the mammalian TGN. In order to further characterise the yeast Golgi, the aim of this project was to prepare vesicles derived specifically from the Kex2p-containing compartment of the yeast Golgi. This aim was pursued in two ways. Firstly, a hybrid protein, consisting of IgG-binding domains of the <i>S.aureus</i> coat protein (protein A) fused to the extreme, cytoplasmically-disposed, C-terminus of Kex2p was constructed. It was intended that IgG-Sepharose would be used to isolate membrane vesicles containing the hybrid protein from yeast cell lysates, and that these vesicles would originate from the Kex2-containing compartment. Unfortunately, this approach was unsuccessful since preliminary results suggest that the protein is mislocalised to the vacuole. The second approach that was employed involved the generation of a polyclonal antibody preparation that specifically recognises the cytoplasmically-disposed C-terminal region of Kex2p. This was achieved using bacterially-produced hybrid proteins (containing the C-terminal 100 residues of Kex2p) to immunise rabbits, and to affinity-purify Kex20 specific antibodies from immune sera.