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A functional connection of Dictyostelium paracaspase with the contractile vacuole and a possible partner of the vacuolar proton ATPase

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Abstract

Dictyostelium discoideum possesses only one caspase family member, paracaspase (pcp). Two separate mutant cell lines were first analysed: one cell line was an over-expressed GFP-tagged Pcp (GFP-Pcp), while the other cell line was a pcp-null (pcp-). Microscopic analysis of cells expressing GFP-Pcp revealed that Pcp was associated with the contractile vacuole membrane consisting of bladder-like vacuoles. This association was disrupted when cells were exposed to osmotic stress conditions. Compared with wild-type cells, the GFP-Pcp-over-expressing cells were susceptible to osmotic stress and were seen to be very rounded in hypo-osmotic conditions and contained more abnormally swollen contractile vacuole. Cells with pcp- were also rounded but had few, if any, contractile vacuoles. These observations suggest that Pcp is essential for Dictyostelium osmotic regulation via its functioning in the contractile vacuole system. Subjecting these cells to selected contractile vacuole inhibitor provided additional support for these findings. Furthermore, yeast two-hybrid system identified vacuolar proton ATPase (VatM) as the protein interacting with Pcp. Taken together, this work gives evidence for an eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the normal morphology of the cell.

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Acknowledgements

We greatly appreciate Dr Pierre Golstein for providing the D. discoideum cells with pcp-null mutant. We gratefully acknowledge Dr Fusheng Tang for providing the pGAD-C3 and pGBD-C3 plasmids and yeast strain PJ69-4A, as well as helpful suggestions about the yeast two-hybrid systems. We thank Dr Maurice Kleve for his expert technical assistance. We also thank Richard Connor for his help in lab work. Finally, we are grateful to Dr Marinelle Ringer for her assistance in proofreading the manuscript.

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  1. Biology Department, University of Arkansas at Little Rock, 2801 South University Ave, Little Rock, AR, 72204-1099, USA

    Entsar Saheb, Ithay Biton, Katherine Maringer & John Bush

  2. Biology Department, Baghdad University, Baghdad, Iraq

    Entsar Saheb

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  1. Entsar Saheb
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  2. Ithay Biton
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  3. Katherine Maringer
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  4. John Bush
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Correspondence to John Bush.

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Corresponding editor: B Jagadeeshwar Rao

[Saheb E, Biton I, Maringer K and Bush J 2013 A functional connection of Dictyostelium paracaspase with the contractile vacuole and a possible partner of the vacuolar proton ATPase. J. Biosci. 38 1–13] DOI 10.1007/s12038-013-9338-3

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Saheb, E., Biton, I., Maringer, K. et al. A functional connection of Dictyostelium paracaspase with the contractile vacuole and a possible partner of the vacuolar proton ATPase. J Biosci 38, 509–521 (2013). https://doi.org/10.1007/s12038-013-9338-3

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