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THE PLANT CELL, Vol 8, Issue 9 1545-1553, Copyright © 1996 by American Society of Plant Biologists
Physical Association of KAB1 with Plant K+ Channel [alpha] Subunits
H. Tang, A. C. Vasconcelos and G. A. Berkowitz
Plant Science Department, Cook College, Rutgers-The State University of New Jersey, New Brunswick, New Jersey 08903
K+ channel proteins contain four [alpha] subunits that align along a
central axis perpendicular to membranes and form an ion-conducting pore.
Recent work with K+ channels native to animal membranes has shown that at
least some members of this protein family also have four [beta] subunits.
These structural components of the holoenzyme each form tight associations
with the cytoplasmic portion of an [alpha] subunit. We have cloned an
Arabidopsis cDNA (KAB1) that encodes a polypeptide sharing 49% amino acid
identity with animal K+ channel [beta] subunits. In this study, we provide
experimental evidence that the KAB1 polypeptide forms a tight physical
association with the Arabidopsis K+ channel [alpha] subunit, KAT1. An
affinity-purified KAB1 fusion protein was immobilized to a support resin
and shown to sequester selectively the KAT1 polypeptide. In addition,
polyclonal antibodies raised against KAB1 were shown to immunoprecipitate
the KAT1 polypeptide as a KAT1-KAB1 protein complex. Immunoblot analysis
demonstrated that KAB1 is expressed in Arabidopsis seedlings and is present
in both membrane and soluble protein fractions. The presence of KAB1 (a
soluble polypeptide) in both soluble and membrane protein fractions
suggests that a portion of the total amount of native KAB1 is associated
with an integral membrane protein, such as KAT1. The presence of KAB1 in
crude protein fractions prepared from different Arabidopsis plant organs
was evaluated. High levels of KAB1 protein were present in flowers, roots,
and leaves. Immunoblot analysis of protein extracts prepared from broad
bean leaves indicated that the KAB1 expression level was 80-fold greater in
guard cells than in mesophyll cells. Previous studies of the in situ
transcription pattern of KAT1 in Arabidopsis indicated that this [alpha]
subunit is abundantly present in leaves and, within the leaf, exclusively
present in guard cells. Thus, KAB1 was determined to be expressed in plant
organs (leaves) and cell types (guard cells) that are sites of KAT1
expression in the plant. The in situ expression pattern of KAB1 suggests
that it may associate with more than one type of K+ channel [alpha]
subunit. Sequence analysis indicates that KAB1 may function in plant K+
channels as an oxidoreductase. It is postulated that [beta] subunits native
to animal K+ channels act as regulatory subunits through pyridine
nucleotide-linked reduction of a [alpha] polypeptides. Although the KAB1
primary structure is substantially different from that of animal [beta]
subunits, amino acid motifs critical for this catalytic activity are
retained in the plant [beta] subunit.
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