Plant Molecular Biology 50: 535–542, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
Post-translational modiﬁcation of barley 14-3-3A is isoform-speciﬁc and
involves removal of the hypervariable C-terminus
, Mieke J. van Zeijl
, Katrine Drumm
, Michael G. Palmgren
, Jan W. Kijne
and Mei Wang
Centre for Phytotechnology, Leiden University/Netherlands Organization for Applied Scientiﬁc Research
(UL/TNO), TNO Department of Applied Plant Sciences, Wassenaarseweg 64, 2333 AL Leiden, Netherlands
author for correspondence; e-mail firstname.lastname@example.org);
Plant Physiology and Anatomy Laboratory, De-
partment of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg
C, Copenhagen, Denmark;
Section for Plant Pathology, Department of Plant Biology, Royal Veterinary and
Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark;
Centre for Phy-
totechnology, UL/TNO, Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 AL
Present address: Department of Plant Physiology, Swammerdam Institute for Life Sciences,
University of Amsterdam, Kruislaan 318, 1098 KW Amsterdam, Netherlands;
These authors contributed equally
to this work
Received 28 August 2001; accepted in revised form 6 March 2002
Key words: 14-3-3 protein, H
-ATPase, Hordeum distichum, post-translational modiﬁcation, proteolytic cleavage
The 14-3-3 protein family is a family of regulatory proteins involved in diverse cellular processes. In a previous
study of regulation of individual 14-3-3 isoforms in the germinating barley embryo, we found that a post-
translationally modiﬁed, 28 kDa form of 14-3-3A was present in speciﬁc cell fractions of the germinated embryo.
In the present study, we identify the nature of the modiﬁcation of 14-3-3A, and show that the 28 kDa doublet is the
result of cleavage of the C-terminus. The 28 kDa forms of 14-3-3A lack ten or twelve amino acid residues at the
non-conserved C-terminus of the protein, respectively. Barley 14-3-3B and 14-3-3C are not modiﬁed in a similar
way. Like the 30 kDa form, in vitro produced 28 kDa 14-3-3A is still capable of binding AHA2 H
-ATPase in an
overlay assay. Our results show a novel isoform-speciﬁc post-translational modiﬁcation of 14-3-3 proteins that is
regulated in a tissue-speciﬁc and developmental way.
The 14-3-3 protein family consists of highly conserved
proteins that are ubiquitously present in eukaryotic
cells. Although the functions of 14-3-3 proteins are
highly diverse, a general mode of action involves the
association with other proteins. Up to now, more than
sixty 14-3-3-interacting proteins have been identiﬁed.
By binding their targets, 14-3-3 proteins are involved
in signalling cascades and compartmentalization of
proteins as well as in regulation of enzymes of the
primary metabolism (Fu et al., 2000; Muslin and Xing,
2000; Roberts, 2000).
In plant cells, the best-studied 14-3-3-interacting
proteins are the plasma membrane (PM) H
and the cytosolic enzyme nitrate reductase (NR).
Whereas association with 14-3-3 activates the H
ATPase, it inhibits NR activity (Bachmann et al.,
1996; Moorhead et al., 1996; Jahn et al., 1997; Oeck-
ing et al., 1997). Another function of plant 14-3-3
proteins is the binding of speciﬁc and general tran-
scription factors, such as Em (Schultz et al., 1998),
TBP and TFIIB (Pan et al., 1999).
Most 14-3-3 targets are bound through a similar
mode of association, which requires phosphorylation
of the target protein (Muslin et al., 1996). Inducible