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Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5.

Jeong JY, Johns J, Sinclair C, Park JM, Rossie S - BMC Cell Biol. (2003)

Bottom Line: Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment.In addition, C-terminal truncation did not prevent further activation by lipid.Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA. jyjeong@purdue.edu

ABSTRACT

Background: Protein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level.

Results: The specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth.

Conclusions: Many characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log phase growth suggests that this enzyme is involved in cell growth or its expression is controlled by metabolic or nutritional signals.

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Expression of HA-Ppt1p as a function of cell growth. (A) An overnight culture of WHT4-1 cells (OD600 nm 5~10) was used to inoculate fresh medium to OD600 nm 0.2 and the culture grown at 30°C. At the indicated times samples were prepared and subjected to immunoblotting using anti-HA antibody. The lower panel shows the bottom half of the same blot probed with anti-Cdc28p antibody. This experiment was performed four times independently with similar results. WT; wild type. (B) The growth curve was plotted based on direct cell counting using a hemocytometer. The relative amount of HA-Ppt1p was obtained by taking the ratio of the band intensity of HA-Ppt1p to Cdc28p. (C) Total RNA was isolated at the indicated time after the inoculation of an overnight culture of W303 cells (OD600 nm 5~10) to OD600 nm 0.2. Twenty micrograms of RNA per sample were resolved and subjected to Northern analysis with a 32P-labeled PPT1 cDNA probe. The lower panel shows the same blot probed for yeast actin mRNA. This experiment was repeated four independent times for both W303 and WHT4-1 cells with similar results.
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Figure 4: Expression of HA-Ppt1p as a function of cell growth. (A) An overnight culture of WHT4-1 cells (OD600 nm 5~10) was used to inoculate fresh medium to OD600 nm 0.2 and the culture grown at 30°C. At the indicated times samples were prepared and subjected to immunoblotting using anti-HA antibody. The lower panel shows the bottom half of the same blot probed with anti-Cdc28p antibody. This experiment was performed four times independently with similar results. WT; wild type. (B) The growth curve was plotted based on direct cell counting using a hemocytometer. The relative amount of HA-Ppt1p was obtained by taking the ratio of the band intensity of HA-Ppt1p to Cdc28p. (C) Total RNA was isolated at the indicated time after the inoculation of an overnight culture of W303 cells (OD600 nm 5~10) to OD600 nm 0.2. Twenty micrograms of RNA per sample were resolved and subjected to Northern analysis with a 32P-labeled PPT1 cDNA probe. The lower panel shows the same blot probed for yeast actin mRNA. This experiment was repeated four independent times for both W303 and WHT4-1 cells with similar results.

Mentions: To examine when Ppt1p is expressed and where it is localized, a yeast strain (WHT4-1) was generated in which the PPT1 gene was replaced with cDNA encoding hemagglutinin-tagged Ppt1p (HA-Ppt1p). Immunoblotting showed that HA-Ppt1p levels were highest in early log phase, and then decreased dramatically during the stationary phase (Fig. 4A,4B). Northern analysis showed that the PPT1 mRNA levels also increased in early log phase and decreased as the cell division decreased upon entering the stationary phase (Fig. 4C), correlating with protein expression. The pattern of PPT1 gene expression was identical in wild-type cells and cells expressing HA-Ppt1p, indicating that the epitope tag did not alter transcription or message stability (data not shown).


Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5.

Jeong JY, Johns J, Sinclair C, Park JM, Rossie S - BMC Cell Biol. (2003)

Expression of HA-Ppt1p as a function of cell growth. (A) An overnight culture of WHT4-1 cells (OD600 nm 5~10) was used to inoculate fresh medium to OD600 nm 0.2 and the culture grown at 30°C. At the indicated times samples were prepared and subjected to immunoblotting using anti-HA antibody. The lower panel shows the bottom half of the same blot probed with anti-Cdc28p antibody. This experiment was performed four times independently with similar results. WT; wild type. (B) The growth curve was plotted based on direct cell counting using a hemocytometer. The relative amount of HA-Ppt1p was obtained by taking the ratio of the band intensity of HA-Ppt1p to Cdc28p. (C) Total RNA was isolated at the indicated time after the inoculation of an overnight culture of W303 cells (OD600 nm 5~10) to OD600 nm 0.2. Twenty micrograms of RNA per sample were resolved and subjected to Northern analysis with a 32P-labeled PPT1 cDNA probe. The lower panel shows the same blot probed for yeast actin mRNA. This experiment was repeated four independent times for both W303 and WHT4-1 cells with similar results.
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Related In: Results  -  Collection

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Figure 4: Expression of HA-Ppt1p as a function of cell growth. (A) An overnight culture of WHT4-1 cells (OD600 nm 5~10) was used to inoculate fresh medium to OD600 nm 0.2 and the culture grown at 30°C. At the indicated times samples were prepared and subjected to immunoblotting using anti-HA antibody. The lower panel shows the bottom half of the same blot probed with anti-Cdc28p antibody. This experiment was performed four times independently with similar results. WT; wild type. (B) The growth curve was plotted based on direct cell counting using a hemocytometer. The relative amount of HA-Ppt1p was obtained by taking the ratio of the band intensity of HA-Ppt1p to Cdc28p. (C) Total RNA was isolated at the indicated time after the inoculation of an overnight culture of W303 cells (OD600 nm 5~10) to OD600 nm 0.2. Twenty micrograms of RNA per sample were resolved and subjected to Northern analysis with a 32P-labeled PPT1 cDNA probe. The lower panel shows the same blot probed for yeast actin mRNA. This experiment was repeated four independent times for both W303 and WHT4-1 cells with similar results.
Mentions: To examine when Ppt1p is expressed and where it is localized, a yeast strain (WHT4-1) was generated in which the PPT1 gene was replaced with cDNA encoding hemagglutinin-tagged Ppt1p (HA-Ppt1p). Immunoblotting showed that HA-Ppt1p levels were highest in early log phase, and then decreased dramatically during the stationary phase (Fig. 4A,4B). Northern analysis showed that the PPT1 mRNA levels also increased in early log phase and decreased as the cell division decreased upon entering the stationary phase (Fig. 4C), correlating with protein expression. The pattern of PPT1 gene expression was identical in wild-type cells and cells expressing HA-Ppt1p, indicating that the epitope tag did not alter transcription or message stability (data not shown).

Bottom Line: Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment.In addition, C-terminal truncation did not prevent further activation by lipid.Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA. jyjeong@purdue.edu

ABSTRACT

Background: Protein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level.

Results: The specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth.

Conclusions: Many characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log phase growth suggests that this enzyme is involved in cell growth or its expression is controlled by metabolic or nutritional signals.

Show MeSH
Related in: MedlinePlus