Figure 5. Dynamics of Inp2p in cells defective in peroxisome transfer to buds. (A and B) Inp2p accumulates on most peroxisomes in mother cells of myo2 mutants defective in peroxisome inheritance. Cells harboring the MYO2 gene (A) or myo2-Y1483A mutation (B) and expressing Inp2p-GFP and peroxisomal mRFP-SKL were grown in YPD to mid-log phase, and confocal images were captured. Left panels show the merged image of the middle and right panels. The red arrow indicates a peroxisome in a mother cell of the MYO2 strain that contains a detectable amount of Inp2p-GFP. Asterisks indicate myo2-Y1483A mutant cells that have delivered peroxisomes to their buds. Bars, 5 µm. (C) Inp2p levels are increased in cells defective in peroxisome inheritance. Cells harboring either MYO2 or the myo2-Y1483A mutant allele and expressing Inp2p-pA were grown in YPD and synchronized in G1 by addition of α factor (0 min). After removal of α factor, cells were incubated at 23°C in YPD. Samples were collected at the times indicated, and total cell lysates were prepared and analyzed by immunoblotting with rabbit IgG to detect Inp2p-pA and antibodies recognizing the cyclin Clb2p or G6PDH. Clb2p monitors progression through the cell cycle. G6PDH serves as a protein loading control. Detection of Inp2p-pA was for short (top) and long (bottom) exposures. The short exposure facilitates the observation of more slowly migrating phosphorylated forms of Inp2p. (D) Inp2p is phosphorylated. Total cell extracts of wild-type cells expressing Inp2p-pA were treated with water (−) or calf intestine alkaline phosphatase (+). Immunoblot analysis was performed using rabbit IgG to detect Inp2p-pA (arrows indicate the more slowly migrating species of Inp2p-pA) and anti-G6PDH antibodies. (C and D) Numbers in parentheses denote the approximate sizes of proteins in kilodaltons. (E) Northern blot analysis of total RNA from MYO2 and myo2-Y1483A strains grown in YPD to exponential phase. Blots were hybridized to probes from the coding regions of the INP2 and ACT1 genes. ACT1 RNA serves as a loading control for total RNA. Numbers in parentheses denote the approximate sizes of transcripts in kilobases.
Figure 5.

Dynamics of Inp2p in cells defective in peroxisome transfer to buds. (A and B) Inp2p accumulates on most peroxisomes in mother cells of myo2 mutants defective in peroxisome inheritance. Cells harboring the MYO2 gene (A) or myo2-Y1483A mutation (B) and expressing Inp2p-GFP and peroxisomal mRFP-SKL were grown in YPD to mid-log phase, and confocal images were captured. Left panels show the merged image of the middle and right panels. The red arrow indicates a peroxisome in a mother cell of the MYO2 strain that contains a detectable amount of Inp2p-GFP. Asterisks indicate myo2-Y1483A mutant cells that have delivered peroxisomes to their buds. Bars, 5 µm. (C) Inp2p levels are increased in cells defective in peroxisome inheritance. Cells harboring either MYO2 or the myo2-Y1483A mutant allele and expressing Inp2p-pA were grown in YPD and synchronized in G1 by addition of α factor (0 min). After removal of α factor, cells were incubated at 23°C in YPD. Samples were collected at the times indicated, and total cell lysates were prepared and analyzed by immunoblotting with rabbit IgG to detect Inp2p-pA and antibodies recognizing the cyclin Clb2p or G6PDH. Clb2p monitors progression through the cell cycle. G6PDH serves as a protein loading control. Detection of Inp2p-pA was for short (top) and long (bottom) exposures. The short exposure facilitates the observation of more slowly migrating phosphorylated forms of Inp2p. (D) Inp2p is phosphorylated. Total cell extracts of wild-type cells expressing Inp2p-pA were treated with water (−) or calf intestine alkaline phosphatase (+). Immunoblot analysis was performed using rabbit IgG to detect Inp2p-pA (arrows indicate the more slowly migrating species of Inp2p-pA) and anti-G6PDH antibodies. (C and D) Numbers in parentheses denote the approximate sizes of proteins in kilodaltons. (E) Northern blot analysis of total RNA from MYO2 and myo2-Y1483A strains grown in YPD to exponential phase. Blots were hybridized to probes from the coding regions of the INP2 and ACT1 genes. ACT1 RNA serves as a loading control for total RNA. Numbers in parentheses denote the approximate sizes of transcripts in kilobases.

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