Skip to main content
PMC home page
The Plant Cell logoLink to The Plant Cell
. 1996 Sep;8(9):1545–1553. doi: 10.1105/tpc.8.9.1545

Physical association of KAB1 with plant K+ channel alpha subunits.

H Tang 1, A C Vasconcelos 1, G A Berkowitz 1
PMCID: PMC161297  PMID: 8837508

Abstract

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 seedings 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 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.

Full Text

The Full Text of this article is available as a PDF (2.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson J. A., Huprikar S. S., Kochian L. V., Lucas W. J., Gaber R. F. Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3736–3740. doi: 10.1073/pnas.89.9.3736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bengal E., Ransone L., Scharfmann R., Dwarki V. J., Tapscott S. J., Weintraub H., Verma I. M. Functional antagonism between c-Jun and MyoD proteins: a direct physical association. Cell. 1992 Feb 7;68(3):507–519. doi: 10.1016/0092-8674(92)90187-h. [DOI] [PubMed] [Google Scholar]
  3. Briskin D. P. Intermediate reaction states of the red beet plasma membrane ATPase. Arch Biochem Biophys. 1986 Jul;248(1):106–115. doi: 10.1016/0003-9861(86)90406-6. [DOI] [PubMed] [Google Scholar]
  4. Brown A. M. Functional bases for interpreting amino acid sequences of voltage-dependent K+ channels. Annu Rev Biophys Biomol Struct. 1993;22:173–198. doi: 10.1146/annurev.bb.22.060193.001133. [DOI] [PubMed] [Google Scholar]
  5. Chouinard S. W., Wilson G. F., Schlimgen A. K., Ganetzky B. A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6763–6767. doi: 10.1073/pnas.92.15.6763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dolly J. O., Rettig J., Scott V. E., Parcej D. N., Wittkat R., Sewing S., Pongs O. Oligomeric and subunit structures of neuronal voltage-sensitive K+ channels. Biochem Soc Trans. 1994 May;22(2):473–478. doi: 10.1042/bst0220473. [DOI] [PubMed] [Google Scholar]
  7. Frangioni J. V., Neel B. G. Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem. 1993 Apr;210(1):179–187. doi: 10.1006/abio.1993.1170. [DOI] [PubMed] [Google Scholar]
  8. Jan L. Y., Jan Y. N. Potassium channels and their evolving gates. Nature. 1994 Sep 8;371(6493):119–122. doi: 10.1038/371119a0. [DOI] [PubMed] [Google Scholar]
  9. Kruse T., Tallman G., Zeiger E. Isolation of Guard Cell Protoplasts from Mechanically Prepared Epidermis of Vicia faba Leaves. Plant Physiol. 1989 Aug;90(4):1382–1386. doi: 10.1104/pp.90.4.1382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Li W., Assmann S. M. Characterization of a G-protein-regulated outward K+ current in mesophyll cells of vicia faba L. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):262–266. doi: 10.1073/pnas.90.1.262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Majumder K., De Biasi M., Wang Z., Wible B. A. Molecular cloning and functional expression of a novel potassium channel beta-subunit from human atrium. FEBS Lett. 1995 Mar 13;361(1):13–16. doi: 10.1016/0014-5793(95)00120-x. [DOI] [PubMed] [Google Scholar]
  12. McCormack K., McCormack T., Tanouye M., Rudy B., Stühmer W. Alternative splicing of the human Shaker K+ channel beta 1 gene and functional expression of the beta 2 gene product. FEBS Lett. 1995 Aug 14;370(1-2):32–36. doi: 10.1016/0014-5793(95)00785-8. [DOI] [PubMed] [Google Scholar]
  13. McCormack T., McCormack K. Shaker K+ channel beta subunits belong to an NAD(P)H-dependent oxidoreductase superfamily. Cell. 1994 Dec 30;79(7):1133–1135. doi: 10.1016/0092-8674(94)90004-3. [DOI] [PubMed] [Google Scholar]
  14. Morales M. J., Castellino R. C., Crews A. L., Rasmusson R. L., Strauss H. C. A novel beta subunit increases rate of inactivation of specific voltage-gated potassium channel alpha subunits. J Biol Chem. 1995 Mar 17;270(11):6272–6277. doi: 10.1074/jbc.270.11.6272. [DOI] [PubMed] [Google Scholar]
  15. Parcej D. N., Scott V. E., Dolly J. O. Oligomeric properties of alpha-dendrotoxin-sensitive potassium ion channels purified from bovine brain. Biochemistry. 1992 Nov 17;31(45):11084–11088. doi: 10.1021/bi00160a018. [DOI] [PubMed] [Google Scholar]
  16. Reid P. F., Pongs O., Dolly J. O. Cloning of a bovine voltage-gated K+ channel gene utilising partial amino acid sequence of a dendrotoxin-binding protein from brain cortex. FEBS Lett. 1992 May 4;302(1):31–34. doi: 10.1016/0014-5793(92)80277-n. [DOI] [PubMed] [Google Scholar]
  17. Ruth P., Röhrkasten A., Biel M., Bosse E., Regulla S., Meyer H. E., Flockerzi V., Hofmann F. Primary structure of the beta subunit of the DHP-sensitive calcium channel from skeletal muscle. Science. 1989 Sep 8;245(4922):1115–1118. doi: 10.1126/science.2549640. [DOI] [PubMed] [Google Scholar]
  18. Schroeder J. I., Ward J. M., Gassmann W. Perspectives on the physiology and structure of inward-rectifying K+ channels in higher plants: biophysical implications for K+ uptake. Annu Rev Biophys Biomol Struct. 1994;23:441–471. doi: 10.1146/annurev.bb.23.060194.002301. [DOI] [PubMed] [Google Scholar]
  19. Scott V. E., Rettig J., Parcej D. N., Keen J. N., Findlay J. B., Pongs O., Dolly J. O. Primary structure of a beta subunit of alpha-dendrotoxin-sensitive K+ channels from bovine brain. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1637–1641. doi: 10.1073/pnas.91.5.1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tang H., Vasconcelos A. C., Berkowitz G. A. Evidence that plant K+ channel proteins have two different types of subunits. Plant Physiol. 1995 Sep;109(1):327–330. doi: 10.1104/pp.109.1.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tymms M. J., McInnes B. Efficient in vitro expression of interferon alpha analogs using SP6 polymerase and rabbit reticulocyte lysate. Gene Anal Tech. 1988 Jan-Feb;5(1):9–15. doi: 10.1016/0735-0651(88)90021-0. [DOI] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES