Figure 3.

Phosphorylation of Hsl1p T273 is important for Hsl1p function. (A) Alignment of Snf1p and Hsl1p activation loop sequences. (B) Hsl1p, Hsl1p^T273A, and Hsl1p^T273E are expressed at similar levels in vivo. Untagged (DLY1), HSL1-myc HSL7-HA (DLY8113), hsl1^T273Amyc HSL7-HA (DLY7841), and HSL1^T273Emyc HSL7-HA (DLY10072) cells were grown to exponential phase at 30°C, lysed by TCA precipitation, and Hsl1p-myc, and Hsl7p-HA were detected by Western blotting. (C) Hsl1p^T273A is less biologically active than Hsl1p or Hsl1p^T273E. HSL1-myc mih1Δ (DLY9882), HSL1^T273Emyc mih1Δ (DLY 11005), hsl1^T273Amyc mih1Δ (DLY7980), HSL1-myc mih1Δ swe1Δ (DLY9883), HSL1^T273Emyc mih1Δ swe1Δ (DLY 11006), and hsl1^T273Amyc mih1Δ swe1Δ (DLY11007) cells were grown to exponential phase at 30°C and examined by DIC microscopy. (D) Cell cycle profiles of the cells from C were determined by scoring the budding and nuclear division status of DAPI-stained cells (n = 250). (E) Hsl1p^T273A is localized to the bud neck, but does not effectively recruit Hsl7p. HSL1-myc HSL7-HA (DLY8113), hsl1^T273Amyc HSL7-HA (DLY7841), HSL1^T273Emyc HSL7-HA (DLY7451), and hsl1^K110Rmyc HSL7-HA (DLY8117) cells were grown to exponential phase at 30°C and processed for immunofluorescence to visualize the septin Cdc11p together with either Hsl1p-myc (top pairs) or Hsl7p-HA (bottom pairs). Representative cells are shown (left) together with the results of scoring 250 cells with a septin collar for the presence of detectable Hsl1p or Hsl7p (right).