Fig. 3. Predicted topology of Ice2p. (A) Ice2p might aggregate at 100°C. Lysates were prepared from strains SFNY1082 (Ice2p-GFP) and NY873 (an untagged control strain). Protein samples were TCA precipitated and subjected to SDS-PAGE and western blot analysis using an affinity-purified polyclonal antibody directed against GFP (lanes 1,2). A band corresponding to Ice2p-GFP that migrated below the 66 kDa marker was found in lane 1 but not in the untagged control (lane 2). Ice2p-GFP was not detected when samples in lanes 3 and 4 (untagged control) were heated for 5 minutes at 100°C in sample buffer. Lysates were also prepared from a strain in which Ice2p was tagged at its C-terminus with a 13x-Myc epitope. Samples were TCA precipitated, resolved on a 10% SDS-polyacrylamide gel and subjected to immunoblot analysis using monoclonal antibodies directed against the Myc epitope on Ice2p. Lane 5 contains Ice2p-Myc. Lane 6 contains a lysate prepared from an untagged strain (NY873). (B) The mobility of Ice2p was not altered upon EndoH treatment. Lysates from the untagged control and SFNY1283 (Ice2p-Myc) were digested with EndoH (lanes 2, 4) for 16 hours. Samples were resolved by SDS-PAGE and subjected to western blot analysis using anti-Myc (B, top) and anti-CPY antibodies (B, bottom). (C) Schematic of Ice2p topology. Putative glycosylation sites lie in the second and third cytosolic loops of Ice2p (asterisks). This analysis supports the hypothesis that Ice2p is a type-III membrane protein.