Plant Molecular Biology 44: 499–511, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
The Arabidopsis thaliana PPX/PP4 phosphatases: molecular cloning and
structural organization of the genes and immunolocalization of the
proteins to plastids
, Tobias I. Baskin
, Antonio Casamayor
, Nuria Cortadellas
, Albert Ferrer
Dept. de Bioqu´ımica i Biologia Molecular, Facultat de Farm`acia, Universitat de Barcelona, 08028 Barcelona,
Division of Biological Sciences, University of Missouri, Columbia, MO, USA;
Dept. Bioqu´ımica i Bi-
olog´ıa Molecular, Facultat de Veterin`aria, Universitat Aut`onoma de Barcelona, 08193 Bellaterra Barcelona, Spain
author for correspondence; e-mail: Joaquin.Arino@uab.es);
Unitat de Microscop´ıa de Transmissi´o, Serveis
Cient´ıﬁco-T`ecnics, Universitat de Barcelona, 08028 Barcelona, Spain
Received 22 October 1999; accepted in revised form 23 June 2000
Key words: gene comparison, PPX-1, PPX-2, protein phosphatase, root plastid, subcellular localization
The PPX/PP4 Ser/Thr protein phosphatases belong to the type 2A phosphatase subfamily and are present in most
eukaryotic organisms. We have previously isolated two closely related DNAs encoding PPX isoforms (PPX-1 and
PPX-2) of Arabidopsis thaliana. Here we report the molecular cloning of the genes encoding these proteins. The
genes PPX-1 and PPX-2 are composed of eight exons and seven introns located at equivalent positions related to
the coding sequences. Whereas the intron-exon organization of the PPX genes is completely different from that
of the PP2A-3/PP2A-4 A. thaliana family, speciﬁc intron-exon boundaries are conserved among PPX genes from
distantly related organisms. Based on GUS expression, both PPX genes show the same spatial and temporal pattern
of expression: they are expressed in all the organs and tissues analyzed, and from the earliest stage of development.
When PPX proteins were localized to the root in semi-thin methacrylate sections by immunoﬂuorescence, staining
was predominantly conﬁned to small organelles, shown to be plastids by co-localization of PPX and ferredoxin.
Interestingly, only some ferredoxin-positive plastids were also PPX-positive, and PPX staining was consistently
brighter in the epidermis. The localization was conﬁrmed with immunogold and electron microscopy. Our results
suggest that, despite its strong sequence conservation, PPX in plants functions differently than in animals.
A major mechanism of cellular regulation is the phos-
phorylation and dephosphorylation of proteins at ser-
ine and threonine residues. The enzymes that dephos-
phorylate these residues are denominated Ser/Thr pro-
tein phosphatases, and their relevance in the control
of many aspects of the physiology and development
in plants is receiving increasing attention (Mackintosh
and Cohen, 1989; Mackintosh et al., 1991; Jagiello
The nucleotide sequence data reported will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession numbers AF030289 (PPX-1) and AF030290 (PPX-2).
et al., 1992; Takeda et al., 1994; Smith et al., 1994,
Smith and Walker, 1996). Ser/Thr phosphatase ac-
tivities were initially classiﬁed, using biochemical
criteria, into four major groups: type 1, 2A, 2B and 2C
(PP1, PP2A, PP2B, and PP2C, respectively), and this
classiﬁcation can be extended to virtually every eu-
karyotic cell (Cohen, 1989). Type 1, 2A and 2B phos-
phatases are oligomeric enzymes and their catalytic
subunits are closely related in sequence, deﬁning a
gene family designated as PPP.
In addition to the so-called ‘classical’ phosphatases
(PP1, PP2A and PP2B), the PPP family includes a
number of forms related to PP1 or PP2A (see Cohen,