Distribution of the LxxQxTG motif in Sla1p and phosphorylation of Sla1p by Prk1p. (A) Schematic diagram of the protein structure of Sla1p. Sla1p contains three SH3 domains (solid boxes) at the N terminus. The C-terminal region contains five copies of the LxxQxTG motif (black-hatched boxes) and nine copies of the QxTG motif (cross-hatched boxes). The positions of each motif are as shown. The arrows below the diagram indicate the regions of Sla1p used in GST-fusion proteins. (B) In vitro phosphorylation of Sla1p by Prk1p. Wild-type Prk1p and the kinase-inactivated Prk1p (Prk1^D158Yp) were expressed as HA-tagged proteins and quantified by immunoprecipitation and Western. Equivalent amounts of the immunoprecipitated proteins were used in the kinase reactions (HA-Prk1p, lane 1–3; HA-Prk1^D158Yp, lane 5). Immunoprecipitates from cells containing untagged Prk1p were used in lane 4 as the control. Substrates used in lanes 1–5 were GST-SH3, GST-SR, GST, GST-SR, and GST-SR, respectively. Phosphorylation results were shown as autoradiography (left) and the input substrates were visualized by the Coomassie Blue staining (right).

Physical associations of Prk1^D158Yp with Sla1p and Pan1p. Yeast extracts prepared from various strains in equal amounts were subjected to anti-Myc or anti-HA immunoprecipitations (IP), gel electrophoresis, and analysis by immunoblotting with the use of anti-Myc and anti-HA antibodies, as indicated. (A) Coimmunoprecipitation of HA-Prk1^D158Yp and Myc-Sla1p. Yeast extracts were prepared from the wild-type strain W303 (lanes 1 and 6), W303 containing pGAL-HA-PRK1^D158Y (lanes 4 and 7), W303 containing pMyc-SLA1 (lane 2), and W303 containing both pGAL-HA-PRK1^D158Y and pMyc-SLA1 (lanes 3, 5, and 8), respectively. (B) Coimmunoprecipitation of HA-Prk1^D158Yp and Pan1p-Myc. Yeast extracts were prepared from W303 (lanes 1 and 6), W303 containing pGAL-HA-PRK1^D158Y (lanes 4 and 7), YMC441 (pan1::PAN1-Myc; lane 2), and YMC441 containing pGAL-HA-PRK1^D158Y (lanes 3, 5, and 8), respectively.

Phosphorylation of Sla1p and Pan1p by their associated kinases. Equal amounts of yeast extracts prepared from various strains were subjected to anti-Myc immunoprecipitation followed by kinase reaction as described in MATERIALS AND METHODS. The samples were electrophoresed and analyzed by autoradiography and immunoblotting. (A) Phosphorylation of Sla1p. Yeast extracts were prepared from W303 (wt, lanes 1 and 2), YMC427 (prk1Δ, lanes 3 and 4), YMC439 (prk1Δ ark1Δ, lanes 5 and 6), and YMC438 (ark1Δ, lanes 7 and 8) containing either pMyc-SLA1 (lanes 1, 3, 5, and 7) or pRS424-SLA1 (lanes 2, 4, 6, and 8), respectively. (B) Phosphorylation of Pan1p. Yeast extracts were prepared from YMC441 (pan1::PAN1-Myc; lane 1), W303 (lane 2), YMC442 (prk1Δ pan1::PAN1-Myc; lane 3), YMC427 (prk1Δ; lane 4), YMC444 (prk1Δ ark1Δ pan1::PAN1-Myc; lane 5), YMC439 (prk1Δ ark1Δ; lane 6), YMC443 (ark1Δ pan1::PAN1-Myc; lane 7), and YMC438 (ark1Δ; lane 8), respectively.

Hindrance of the Pan1p/Sla1p interaction by Prk1p-dependent phosphorylation. (A) Phosphorylation of LR1 by Prk1p in vivo. Yeast extracts in equal amounts prepared from W303 containing pRS316 and pRS315 (lane 1), pGAL-HA-LR1 and pRS315 (lane 2), pGAL-HA-LR1 and pGAL-PRK1^D158Y (lane 3), and pGAL-HA-LR1 and pGAL-PRK1 (lanes 4, 5, and 6) were subjected to anti-HA immunoprecipitation and immunoblotting. In lane 5, the immunoprecipitates were treated with CIP before electrophoresis. In lane 6, the yeast extracts were incubated with CIP before immunoprecipitation. (B) Binding of HA-LR1 to GST-SR. Yeast extracts in the same amounts as used for anti-HA immunoprecipitation prepared from W303 containing pGAL-HA-LR1 and pRS315 (lanes 1 and 2), pGAL-HA-LR1 and pGAL-PRK1^D158Y (lane 3), and pGAL-HA-LR1 and pGAL-PRK1 (lanes 4 and 5) were incubated with immobilized GST (lane 1), and GST-SR (lanes 2–5), respectively. The precipitates were separated by SDS-PAGE and immunoblotted with anti-HA antibody. In lane 5, the yeast extracts were treated with CIP before incubation with GST-SR. (C) Phosphorylation of SR by Prk1p in vivo. Equal amounts of yeast extracts prepared from W303 containing pRS316 and pRS315 (lane 1), pGAL-HA-SR and pRS315 (lane 2), pGAL-HA-SR and pGAL-PRK1^D158Y (lane 3), and pGAL-HA-SR and pGAL-PRK1 (lanes 4 and 5) were subjected to anti-HA immunoprecipitation and immunoblotting. In lane 5, the immunoprecipitates were treated with CIP before electrophoresis. HA-SR in the yeast extracts treated with CIP before IP was not presented because it got mostly degraded after the treatment. (D) Binding of HA-SR to GST-LR1. Yeast extracts in the same amounts as used for anti-HA immunoprecipitation prepared from W303 containing pGAL-HA-SR and pRS315 (lanes 1 and 2), pGAL-HA-SR and pGAL-PRK1 (lane 3), and pGAL-HA-SR and pGAL-PRK1^D158Y (lane 4) were incubated with immobilized GST (lane 1) and GST-LR1 (lanes 2–4), respectively. The precipitates were separated by SDS-PAGE and immunoblotted with anti-HA antibody.

Dissociation of Pan1p and Sla1p in PRK1 overexpressing Cells. Yeast extracts prepared from YMC440 (pan1::PAN1-HA) containing pMyc-SLA1 and pRS315 (A), pMyc-SLA1 and pGAL-PRK1 (B), and pMyc-SLA1 and pGAL-PRK1^D158Y (C), were fractionated by gel filtration on a Superose 6 column as described in MATERIALS AND METHODS. Fractions were examined for the presence of Pan1p-HA and Myc-Sla1p. The column was precalibrated with the molecular mass markers as indicated. BD, blue dextran; TG, thyroglobulin; BA, β-amylase; AD, alcohol dehydrogenase; CA, carbonic anhydrase.

Disruption of the colocalization of Pan1p and Sla1p by PRK1 overexpression. YMC445 (pan1::PAN1-RFP sla1::SLA1-GFP) containing pRS316 (A), pGAL-PRK1 (B), and pGAL-PRK1^D158Y (C) were grown to the log phase in medium without galactose (Gal, 0 h) or with galactose (Gal, 2 h). Cells were fixed and visualized for Pan1-RFP (left), Sla1-GFP (middle), and Nomarski image (right) by Leica DMRXA microscope. Bar, 5 μm.

Hindrance of the Pan1p/End3p interaction by Prk1p-dependent phosphorylation. (A) Phosphorylation of LR2 by Prk1p in vivo. Yeast extracts in equal amounts prepared from W303 containing pRS316 and pRS315 (lane 1), pGAL-HA-LR2 and pRS315 (lane 2), pGAL-HA-LR2 and pGAL-PRK1^D158Y (lane 3), and pGAL-HA-LR2 and pGAL-PRK1 (lanes 4, 5, and 6) were subjected to anti-HA immunoprecipitation and immunoblotting. In lane 5, the immunoprecipitates were treated with CIP before electrophoresis. In lane 6, the yeast extracts were incubated with CIP before immunoprecipitation. (B) Binding of HA-LR2 to GST-END3. Yeast extracts in the same amounts as used for anti-HA immunoprecipitation prepared from W303 containing pGAL-HA-LR2 and pRS315 (lanes 1 and 2), pGAL-HA-LR1 and pGAL-PRK1^D158Y (lane 3), and pGAL-HA-LR1 and pGAL-PRK1 (lanes 4 and 5) were incubated with immobilized GST (lane 1) and GST-END3 (lanes 2–5), respectively. The precipitates were separated by SDS-PAGE and immunoblotted with anti-HA antibody. In lane 5, the yeast extracts were treated with CIP before incubation with GST-SR.

Phenotypic analysis of cells overexpressing PRK1. (A) LY accumulation in wild-type strain W303 containing pRS315 (left), pGAL-PRK1 (middle), and pGAL-PRK1^D158Y (right). Cells were incubated with LY at 30°C for 2 h and visualized with fluorescein isothiocyanate fluorescence optics (top) and phase-contrast optics (bottom). Bar, 5 μm. (B) Internalization of uracil permease in W303 containing pRS315 (▴), pGAL-PRK1 (♦), and pGAL-PRK1^D158Y (●). The uracil permease assays were performed as described in MATERIALS AND METHODS. On addition of cycloheximide (CHX), uracil permease levels at the cell surface decreased. Transport of [¹⁴C]uracil into yeast cells was used as a measurement for the relative amounts of uracil permease retained on the cell surface. Uracil uptake is plotted as the percentage activity relative to the initial time point. The results shown are the averages of two independent assays. (C) Cell wall morphology of W303 containing pRS315 (a and b), pGAL-PRK1 (c and d), and pGAL-PRK1^D158Y (e and f). After exposure to galactose for 5 h, cells were prefixed and processed for electron microscopy with the use of potassium permanganate fixation to enhance visualization of the cell wall and membranes. The arrows in panels c and d indicate the aberrant cell walls which are thicker and consist of more than one layer. Bars, 500 nm.