Journal of Experimental Botany, Vol. 52, No. 361, pp. 1739-1740,
August 1, 2001
© 2001 Oxford University Press
Gene Note |
ZMPP2, a novel type-2C protein phosphatase from maize
1 Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA
2 Pioneer Hi-Bred International, Inc., Johnston, Iowa 50131-1004, USA
3 USDA, Agricultural Research Service, Plant Genetics Research Unit, Columbia, Missouri 65211, USA
Received 5 March 2001; Accepted 30 April 2001
Abstract
A cDNA clone was selected as a candidate for the catalytic subunit of phospho-pyruvate dehydrogenase phosphatase (PDP) by screening a Zea mays expressed sequence tag database with the bovine PDP deduced amino acid sequence. Both strands of the cDNA were completely sequenced. The maize clone contains an open reading frame of 1098 base pairs that encodes a polypeptide of 40 127 Da, ZMPP2. The deduced amino acid sequence of ZMPP2 contains the five PP2C signature domains, as does PDP. However, the expression pattern of ZMPP2, determined by reverse transcriptase-polymerase chain reaction, was different from those of the maize pyruvate dehydrogenase E1
subunit and pyruvate dehydrogenase kinase. Additionally, the predicted subcellular location of ZMPP2 is cytoplasmic, while the pyruvate dehydrogenase complex, regulated by reversible phosphorylation, is mitochondrial. Thus, ZMPP2 is a PP2C-type protein phosphatase related to but distinct from PDP.
Key words: Zea mays, PP2C, phosphatase.
Protein phosphatases comprise a broad group of enzymes that catalyse the hydrolytic cleavage of phosphate from proteins. In conjunction with protein kinases, these enzymes regulate diverse cellular processes ranging from signal transduction to metabolic flux (Rodriguez, 1998
). Members of the PP2C family are phospho-serine/threonine phosphatases that dephosphorylate the alpha subunit of phosphorylase kinase and are uninhibited by the thermostable inhibitors 1 and 2. The PP2C family is unique in that its members require magnesium or manganese for activity, and are insensitive to okadaic acid, microcystin, and calyculin A (Smith and Walker, 1996; Rodriguez, 1998). They comprise only a minor proportion of total phospho-protein phosphatase activity in plant cells (MacKintosh et al., 1991). Except for phospho-pyruvate dehydrogenase phosphatase (PDP), members of the PP2C family are monomeric, lacking regulatory subunits, and do not seem evolutionarily related to the heteromeric phospho-protein phosphatases (Smith and Walker, 1996; Rodriguez, 1998). Members of the PP2C family have a conserved catalytic region, and N-terminal extensions of variable length and sequence.
Perhaps the best characterized PP2C from plants is the phospho-pyruvate dehydrogenase phosphatase (PDP), which regulates flux through the mitochondrial pyruvate dehydrogenase complex (PDC) (Teague et al., 1982; Miernyk and Randall, 1987). In an effort to identify maize PDP, the bovine PDP catalytic subunit deduced amino acid sequence (Lawson et al., 1993) (GenBank Accession number L18966) was used as the query in a BLAST search (Basic Local Alignment Search Tool) (Altschul et al., 1990) of the Pioneer Hi-Bred International expressed sequence tag (EST) database. The sequence reported herein is 1459 bp including a 956 bp open reading frame, and is the longest of a group of contiguous cDNAs. Automated sequencing was performed at the University of Missouri DNA Core facility. Sequencing revealed an ATG initiation codon at nucleotide 123 and an in-frame stop codon at nucleotide 1221. The reading frame encodes a 366 amino acid protein, ZMPP2, of 40 127 Da with a calculated pI of 4.6. The deduced amino acid sequence contains the five signature domains of the PP2C class of protein phosphatases (Smith and Walker, 1996).
The in silico localization of ZMPP2 was predicted using the PSORT (Nakai and Horton, 2000), Predator (http://www.inra.fr/Internet/Produits/Predator/index.html), Target P (Emanuelsson et al., 2000), and MITOPROT (Claros and Vincens, 1996) algorithms. The most likely cellular location is cytoplasmic.
Expression of the ZMPP2 mRNA in various maize organs was determined using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) (Fig. 1). Husks, silks, anthers, and ear shoots were harvested from mature glass house-grown maize plants (Zea mays L., B73, Illinois Seed Foundation, Urbana, IL). Seeds were imbibed overnight and etiolated shoots were harvested from plants grown at 30 °C for 5 d. Roots and leaves were harvested from plants grown at 30 °C with a 10 h photoperiod. Total RNA isolation, DNase treatment, and RT-PCR were accomplished as previously described (Thelen et al., 1999). An Access RT-PCR System (Promega, Madison, WI) was used for 40 cycles, starting with approximately 20 ng of RNA per 10 µl reaction. All oligonucleotides were prepared by the University of Missouri DNA Core facility. For ZMPP2, the oligonucleotides used were: 5'-GCATTGAAGCTTTCATTACTATATCTGCTTGTCAGAGC-3' and 5'-AGTAGCGGATCCATGGTGATTTACCTTAGCAC-3'. mRNA expression patterns of various maize PDC subunits were also determined. The oligonucleotides used for the PDC subunits have been previously described (Thelen et al., 1998, 1999).
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Expression of ZMPP2 was relatively high in roots and etiolated shoots, and low or absent in husks. The RT-PCR patterns for the maize PDC subunits, pyruvate dehydrogenase alpha subunit (E1
) and pyruvate dehydrogenase kinase 1 (PDK1), were well co-ordinated, as would be expected for components of a multi-enzyme complex. As previously reported (Thelen et al., 1998), PDK2 has relatively high expression only in leaves. The RT-PCR patterns presented herein are in good agreement with previous results (Thelen et al., 1998, 1999). In contrast, the expression pattern of ZMPP2 does not follow those of any of the PDC subunits (Fig. 1
).
A BLAST search using the ZMPP2 deduced amino acid sequence as the query revealed homology with PP2C-type protein phosphatases from a wide range of different organisms. The greatest similarity to ZMPP2 was seen with the deduced amino acid sequence of two PP2C-like proteins from Arabidopsis thaliana (accessions CAB79904 and T06308). Similarity was also seen between ZMPP2 and a membrane-associated PP2C protein from Paramecium tetraurelia (accession A55804) (Klumpp et al., 1994), although sequence homology was confined to the five PP2C signature domains. The function of these domains is uncertain due to the paucity of structure/ function analyses that have been performed to date. Thus, the maize PP2C that has been analysed here does not appear to be PDP, but rather a constitutively expressed PP2C protein of unknown function similar to those found in organisms ranging from protozoans to man.
Acknowledgments
The authors are grateful to Nancy R David for her assistance. This research was supported by NSF (IBN-98876680), the MoAES, and the Food for the 21st Century Program. This is Journal Report 12 972 from the Missouri Agricultural Experiment Station. The sequences reported in herein will appear in GenBank under the accession AF213455.
Notes
4 Present address: Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI 48824, USA. 
5 To whom correspondence should be addressed. Fax: +1 573 882 5635. E-mail: randalld{at}missouri.edu
Abbreviations
PDC, pyruvate dehydrogenase complex; PP2C, protein phosphatase type-2C; RT-PCR, reverse transcriptase-polymerase chain reaction; ZMPP2, Zea mays protein phosphatase 2C..
References
Altschul SF, Gish W, Miller W, Myers, EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215, 403–410.[Web of Science][Medline]
Claros MG, Vincens P. 1996. Computational method to predict mitochondrially imported proteins and their targeting sequences. European Journal of Biochemistry 241, 779–786.[Web of Science][Medline]
Emanuelsson O, Nielsen H, Brunak S, von Heijne G. 2000. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. Journal of Molecular Biology 300, 1005–1016.[Web of Science][Medline]
Klumpp S, Hanke C, Donella-Deana A, Beyer A, Kellner R, Pinna LA, Schultz JE. 1994. A membrane-bound protein phosphatase type 2C from Paramecium tetraurelia. Purification, characterization and cloning. Journal of Biological Chemistry 269, 32774–32780.
Lawson JE, Niu XD, Browning KS, Trong HL, Yan J, Reed LJ. 1993. Molecular cloning and expression of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase and sequence similarity with protein phosphatase 2C. Biochemistry 32, 8987–8993.[Medline]
MacKintosh C, Coggins J, Cohen P. 1991. Plant protein phosphatases. Biochemical Journal 273, 733–738.
Miernyk JA, Randall DD. 1987. Some properties of pea mitochondrial phospho-pyruvate dehydrogenase phosphatase. Plant Physiology 83, 311–315.
Nakai K, Horton P. 1999. PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends in Biochemical Sciences 24, 34–36.[Web of Science][Medline]
Rodriguez PL. 1998. Protein phosphatase 2C (PP2C) function in higher plants. Plant Molecular Biology 38, 919–927.[Web of Science][Medline]
Smith RD, Walker JC. 1996. Plant protein phosphatases. Annual Review of Plant Physiology and Plant Molecular Biology 47, 101–125.[Web of Science]
Teague WM, Pettit FH, Wu T, Silberman SR, Reed LJ. 1982. Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney. Biochemistry 21, 5585–5592.[Medline]
Thelen JJ, Miernyk JA, Randall DD. 1999. Molecular cloning and expression analysis of the mitochondrial pyruvate dehydrogenase from maize. Plant Physiology 119, 635–643.
Thelen JJ, Muszynski MG, Miernyk JA, Randall DD. 1998. Molecular analysis of two pyruvate dehydrogenase kinases from maize. Journal of Biological Chemistry 273, 26618–26623.
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