The subcellular localization of plant protein phosphatase 5 isoforms is determined by alternative splicing.
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.
de la Fuente van Bentem
Sergio
S
Plant Pathology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, P.O. Box 94062, 1090 GB Amsterdam, The Netherlands.
Vossen
Jack H
JH
Vermeer
Josephus E M
JE
de Vroomen
Marianne J
MJ
Gadella
Theodorus W J
TW
Haring
Michel A
MA
Cornelissen
Ben J C
BJ
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics*; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology
D007527:Isoenzymes; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; C089641:protein phosphatase 5; D005966:Glucuronidase