Figure 3.
Mlp1p is peripherally associated with NEs but only partially cofractionates with NPCs. Yeast cells were fractionated to produce nuclei (Nuclear Prep, fractions 1–8), NEs (NE prep, fractions 9–12) and NPCs (NPC Prep, fractions 9–17; Rout and Kilmartin, 1990; Rout and Blobel, 1993; Strambio-de-Castillia et al., 1995). Peripherally associated NE components were stripped from the NE fraction by heparin-extraction (NE Prep, fractions 13–16; Strambio-de-Castillia et al., 1995). Proteins from each subcellular fraction were resolved on SDS-PAGE, transferred to nitrocellulose and immunoblotted with the anti-Mlp1p mAb148G11. Fractions that belong to each of the enrichment steps are grouped as indicated by brackets at the top of the gels. Figures on top of the gels refer to the fraction numbers as previously described (Rout and Blobel, 1993; Strambio-de-Castillia et al., 1995). The total spheroplasts lysate (Cells) and subsequent NE- and NPC-containing fractions are indicated on top of each gel. In all cases C- stands for Crude and E- stands for Enriched. H-NEs denotes heparin-extracted NEs. A small arrow points to a band that disappears together with Mlp1p in strains harboring a deletion of the gene and is therefore an Mlp1p breakdown product. Numbers at the bottom of the gels (% Total Cellular Amount) represent an estimate of the amount of Mlp1p specific signal present in each fraction based on quantitative immunoblotting, expressed as a percentage of the total cellular amount. The figures below the bottom brackets (Loading Equivalents) indicate the number of cell equivalents (n) that were used as the starting material to prepare each fraction (Rout and Blobel, 1993; Strambio-de-Castillia et al., 1995).