Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology

Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36 K, that possesses a mitochondrial targeting signal and two transmembrane se gments which are thought to anchor this protein to the outer membrane of th e mitochondrial envelope of infected plant cells, To determine the topology of the virus protein inserted in the cell membrane, as well as the sequenc e requirements for targeting and insertion, an in vivo system was set up in which this could be analysed in the absence of productive virus infection, The 36K protein was expressed in the yeast Saccharomyces cerevisiae in nat ive form or fused to the green fluorescent protein, Using a fluorescence mi croscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, whic h is specific for mitochondria, These aggregates were shown by electron mic roscopy to be composed of either mitochondria or membranes, The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein, Immunoelectron mic roscopy demonstrated that the viral protein is present in the anomalous agg regates and Western blot analysis of protein extracts showed 36K to be resi stant to alkaline, urea or salt extraction, a property of integral membrane proteins.