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CELL BIOLOGY AND SIGNAL TRANSDUCTION

The Subcellular Localization of Plant Protein Phosphatase 5 Isoforms Is Determined by Alternative Splicing^[w]

Plant Pathology (S.d.l.F.v.B., J.H.V., M.J.d.V., B.J.C.C.), Molecular Cytology (J.E.M.V., T.W.J.G.), and Plant Physiology (M.A.H.), Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, P.O. Box 94062, 1090 GB Amsterdam, The Netherlands

Protein serine/threonine phosphatase 5 (PP5) plays an important role in signal transduction in animal cells, but in plants, knowledge about PP5 is scarce. Here, we describe the isolation of a full-length cDNA encoding tomato (Lycopersicon esculentum) PP5 (LePP5) and its expression in Escherichia coli. Biochemical characterization showed that recombinant LePP5 has a low intrinsic protein phosphatase activity. This activity was increased 6- to 10-fold by either removal of the N-terminal tetratricopeptide repeat domain or by addition of fatty acids, indicating that biochemical features specific for PP5 homologs from other species are conserved in tomato. The single-copy LePP5 gene was cloned and shown to encode two mRNA species that arise by alternative pre-mRNA splicing. Similarly, Arabidopsis was found to express two PP5 transcripts, suggesting that alternative splicing of PP5 pre-mRNA is not specific for tomato. Alternative splicing results in a larger transcript containing an additional exon encoding two putative transmembrane domains and, hence, in a larger PP5 isoform. Subcellular fractionation studies on tomato protein lysates indicated that the majority of the 55-kD LePP5 isoform is soluble, whereas the 62-kD isoform is an integral membrane protein. Production of yellow fluorescent protein-PP5 chimeras in plant cells indicated that the 55-kD isoform is localized in both the nucleus and the cytoplasm, whereas the 62-kD isoform is targeted to the endoplasmic reticulum, including the nuclear envelope. Our findings show that alternative splicing generates two LePP5 isoforms with a different subcellular localization.

^[w] The online version of this article contains Web-only data.

^* Corresponding author; e-mail cornelissen{at}science.uva.nl; fax 31–0–20–5257934.

Received May 9, 2003; returned for revision June 24, 2003; accepted July 8, 2003.

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