Figure S4.
Recombinant Cdc14 carries phosphatase activity in vitro. (A) Schematic of the colorimetric phosphatase activity measured spectrophotometrically at 410 nm by determining the amount of pNP produced after the hydrolysis of Pi from pNPP. The addition of NaOH after a specified assay time (e.g., 10 min) serves to stop the phosphatase reaction while simultaneously converting the product p-nitrophenol into the yellow-colored p-nitrophenolate (λmax = 410 nm). (B) The indicated proteins were prepared from E. coli, as detailed in Materials and methods, for recombinant protein expression and purification and analyzed by SDS-PAGE followed by Coomassie Blue staining. (C) Phosphatase activity of recombinant 6His-3FLAG-Cdc14 protein with indicated concentrations was assayed as in A. Shown are the average and standard deviation, from three replication experiments, of p-nitrophenolate absorbance at 405 nm, plot to incubation time. (D) Phosphatase activity of 40 µg/ml recombinant 6His-3FLAG-Cdc14, 40 µg/ml 6His-3FLAG-Cdc14m, or 0.4 µg/ml λ phosphatase assayed in the presence or absence of PI as in A, shown as C. Note that PI almost abolished phosphatase activity in 6His-3FLAG-Cdc14 and λ phosphatase. (E) Phosphatase activity of recombinant 6His-3FLAG-Cdc14 and 6His-3FLAG-Cdc14m (enzymatically dead Cdc14 mutant, Cdc14-C283S) protein was assayed for 30 min as in A. Shown are the average and standard deviation, from three replication experiments, of p-nitrophenolate absorbance at 405 nm, plot to protein concentration. Note that 6His-3FLAG-Cdc14m yielded no detectable absorbance over the background. (F) IP (α-FLAG) of Atg1 (FLAG-Atg1-as) complex from cell lysates derived from the FLAG-Atg1-as cells arrested at prophase I. After washing, the Atg1 (FLAG-Atg1-as) complex was eluted from the resin with FLAG peptide. Eluted proteins were resolved by SDS-PAGE and visualized by Sypro Ruby staining. IB confirmed Flag-Atg1-as protein identity. *, IgG. (G) Protein domain organization of Atg13 from various species, with the location of protein domains annotated by residue number. Highlighted are the Serine-Proline (SP, in red) and Proline-x-Leucine (PxL, light blue box) motif. HORMA, Hop1p, Rev7p, and MAD2. Note that IDR harbors at least one PxL motif and the majority of SP sites. A.U., arbitrary unit. Source data are available for this figure: SourceData FS4.

Recombinant Cdc14 carries phosphatase activity in vitro. (A) Schematic of the colorimetric phosphatase activity measured spectrophotometrically at 410 nm by determining the amount of pNP produced after the hydrolysis of Pi from pNPP. The addition of NaOH after a specified assay time (e.g., 10 min) serves to stop the phosphatase reaction while simultaneously converting the product p-nitrophenol into the yellow-colored p-nitrophenolate (λmax = 410 nm). (B) The indicated proteins were prepared from E. coli, as detailed in Materials and methods, for recombinant protein expression and purification and analyzed by SDS-PAGE followed by Coomassie Blue staining. (C) Phosphatase activity of recombinant 6His-3FLAG-Cdc14 protein with indicated concentrations was assayed as in A. Shown are the average and standard deviation, from three replication experiments, of p-nitrophenolate absorbance at 405 nm, plot to incubation time. (D) Phosphatase activity of 40 µg/ml recombinant 6His-3FLAG-Cdc14, 40 µg/ml 6His-3FLAG-Cdc14m, or 0.4 µg/ml λ phosphatase assayed in the presence or absence of PI as in A, shown as C. Note that PI almost abolished phosphatase activity in 6His-3FLAG-Cdc14 and λ phosphatase. (E) Phosphatase activity of recombinant 6His-3FLAG-Cdc14 and 6His-3FLAG-Cdc14m (enzymatically dead Cdc14 mutant, Cdc14-C283S) protein was assayed for 30 min as in A. Shown are the average and standard deviation, from three replication experiments, of p-nitrophenolate absorbance at 405 nm, plot to protein concentration. Note that 6His-3FLAG-Cdc14m yielded no detectable absorbance over the background. (F) IP (α-FLAG) of Atg1 (FLAG-Atg1-as) complex from cell lysates derived from the FLAG-Atg1-as cells arrested at prophase I. After washing, the Atg1 (FLAG-Atg1-as) complex was eluted from the resin with FLAG peptide. Eluted proteins were resolved by SDS-PAGE and visualized by Sypro Ruby staining. IB confirmed Flag-Atg1-as protein identity. *, IgG. (G) Protein domain organization of Atg13 from various species, with the location of protein domains annotated by residue number. Highlighted are the Serine-Proline (SP, in red) and Proline-x-Leucine (PxL, light blue box) motif. HORMA, Hop1p, Rev7p, and MAD2. Note that IDR harbors at least one PxL motif and the majority of SP sites. A.U., arbitrary unit. Source data are available for this figure: SourceData FS4.

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