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. 1999 Jul;10(7):2265–2283. doi: 10.1091/mbc.10.7.2265

Figure 3.

Figure 3

Expression and temperature growth profile of Sla2p deletion proteins. (A) C-terminal deletions. Crude extracts were prepared from wild-type (full-length Sla2p; DDY426) and mutant cells containing the Sla2p C-terminal deletions Sla2Δ768–968 (DDY1194), Sla2Δ501–968 (DDY1195), and Sla2Δ360–968 (DDY1196), grown at 25°C, and then shifted to 37°C for 3 h. Equal cell equivalents (based on A600 absorbance) were immunoblotted using the N-terminal Sla2p antibody. Cells expressing wild-type or Sla2p C-terminal deletion mutants were then replica plated and grown for 36 h (37 and 34°C), 48 h (30 and 25°C), 72 h (20°C), or 5 d (14°C) on YPD plates before being photographed. (B) N-terminal and central coiled-coil domain deletions. Crude extracts were prepared from wild-type (DDY426) cells containing pRS313 (empty vector) and sla2Δ (DDY540) cells containing pDD367 (Sla2Δ33–359), pDD364 (Sla2Δ33–501), pDD362 (Sla2Δ33–750), pDD371(Sla2Δ360–575), pDD369(Sla2Δ33–359, 576stop), or vector alone, grown at 25°C, and then shifted to 37°C for 3 h. Equal cell equivalents from the 25°C cultures or the 37°C-shifted cultures were immunoblotted using the C-terminal Sla2p antibody, except for extracts containing pDD369, in which case the N-terminal Sla2p antibody was used. Wild-type and sla2Δ cells containing vector alone or expressing these Sla2p deletion mutants were then replica plated and grown for 2 d (37 and 34°C), 3 d (30 and 25°C), 5 d (20°C), or 8 d (14°C) on SC-his plates before being photographed as shown. The first spot of each panel represents growth with a low-copy-number plasmid, and the second spot represents growth with a high-copy-number plasmid. (C) Sla2Δ33–750 overexpression is recessive-lethal. Wild-type and sla2Δ cells expressing either Sla2Δ33–750 under the control of the galactose-inducible GAL1 promotor or containing vector alone (pYES-R) were grown on glucose- or galactose-containing media at 25 or 37°C for 72 h before being photographed. (1) Wild-type cells expressing vector only (pYES-R). (2) Wild-type cells containing galactose-inducible Sla2Δ33–750. (3) sla2Δ cells containing vector only. (4) sla2Δ cells expressing galactose-inducible Sla2Δ33–750.