Figure 4

Subcellular localisation of Nar1p. (A) In situ localisation of Nar1p. Wild-type cells (strain PSY581) were transformed with the high-copy expression vector p426 containing NAR1 or no gene. Log-phase cells were fixed with 2.4% (w/v) formaldehyde, permeabilised and labelled with purified anti-Nar1p (Nar1p) or monoclonal anti-Pgk1p (Pgk1p) antibodies, followed by fluorophore-conjugated secondary antibodies (green). DNA was counterstained with DAPI and is indicated in red. Similar results with low fluorescence intensity were obtained with NAR1 inserted into low-copy expression vector p416MET25. (B) Immunoblot analysis of Nar1p in cell fractions. Log-phase wild-type cells were converted into spheroplasts (Sph), subjected to Dounce homogenisation and, after removal of intact cells and cell debris, the extract was fractionated into post-mitochondrial supernatant (PMS) and crude mitochondria (CM) by centrifugation for 10 min at 12 000 g. A similar fractionation was performed with cells overproducing Nar1p from vector p426-NAR1. A separate wild-type yeast culture was used to isolate nuclei (Nuc) by Ficoll gradient centrifugation (Aris and Blobel, 1991). Equal amounts of protein (20 g/per lane) were separated by SDS–PAGE, blotted and immunostained for Nar1p or marker proteins of known cellular localisation (left panel; cytosolic phosphoglycerate kinase Pgk1p; mitochondrial ATP/ADP carrier Aac2p; endoplasmic reticulum translocon subunit Sec61p; and nuclear DNA polymerase-associated Pol30p). The crude mitochondrial fraction was further separated on a Nycodenz step-gradient into fractions containing enriched mitochondria (Mit) and microsomal membranes (Mem). Samples were analysed by immunostaining (right panel; Mge1p; mitochondrial matrix).