Structural and functional characterization of the nodulin 26 ion channel and the calmodulin-like domain protein kinase from soybean root nodules

Author

Charles David Weaver

Date of Award

5-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Daniel M. Roberts

Committee Members

John Koontz, Gary Stacey, Wesley D. Wicks

Abstract

Nodulin 26 is a nodule-specific integral membrane protein proposed to be found in the symbiosome membrane of nitrogen- fixing soybean root nodules (Fortin et al., 1987; Sandal and Marker, 1988). In this study, nodulin 26 protein has been detected in nitrogen-fixing soybean root nodules by using specific immunological probes. These probes have been utilized to verify that the nodulin 26 protein is specifically expressed in nodule tissue. Furthermore, immunoelectron microscopy, as well as Western blot analysis of isolated membrane fractions, have been used to demonstrate that nodulin 26 is found only in the SM of the infected cells of nitrogen-fixing nodules. In soybean nodules that are infected by mutant rhizobium that are defective in release into symbiosomes, nodulin 26 was expressed at levels comparable to wild-type nodules. Taken together, these data demonstrate that nodulin 26 is a nodule-specific protein that is localized to the symbiosome membrane and that the control of nodulin 26 expression is not contingent upon the release of bacteria into symbiosomes nor upon the establishment of a nitrogen-fixing symbiosis.

Soybean root nodules were found to contain a calcium- dependent protein kinase activity that phosphorylated a synthetic peptide (CK-15) containing a sequence found within the 14 carboxyl terminal residues of nodulin 26. This protein kinase activity was purified from the soluble fraction of nodules. A similar activity was partially purified from symbiosome membranes. The soluble protein kinase was activated by µM Ca²⁺. Neither maximal activity nor the ability to respond to Ca²⁺ were dependent on calmodulin or lipid effectors. These observations suggest that this enzyme is a member of the calmodulin-like domain protein kinase family. This is supported by Western blot analyses that show that the nodule protein kinase crossreacts with monoclonal antibodies against the calmodulin-like domain protein kinase. The symbiosome membrane-associated kinase was resistant to removal by chaotropic agents, such as KI, that are known to remove extrinsic membrane proteins. However, the activity could be solubilized by the zwitterionic detergent CHAPS and displayed 400-fold Ca²⁺-stimulation after chromatography by Mono Q ion-exchange. Unlike the soluble protein, the symbiosome membrane-associated protein kinase did not react with monoclonal antibodies against the calmodulin-like domain protein kinase. Thus, the symbiosome membrane-associated protein kinase appears to be distinct from the soluble protein kinase and is integrally associated with the symbiosome membrane.

Ca²⁺-dependent nodulin 26 phosphorylation was detected in symbiosome membranes in vitro and in intact nodules metabolically-labeled with ^32p-phosphate. The phosphorylation site of nodulin 26 was determined to be a single serine residue, ser²⁶², found in the carboxyl terminus of the molecule. This site was also found within the CK-15 sequence. These data demonstrate that soybean root nodules contain protein kinase activities that are activated by physiologically relevant Ca²⁺ concentrations. Furthermore, these data demonstrate that nodulin 26 is phosphorylated on a single serine residue by a calcium-dependent symbiosome membrane-associated protein kinase. Therefore, nodulin 26 may be a target of Ca²⁺-dependent regulation in soybean root nodules.

Observations that nodulin 26 phosphorylation correlates with malate transport rates in intact symbiosomes led to a direct investigation of the functional activity of nodulin 26. To accomplish this, nodulin 26 was purified to apparent homogeneity from soybean root nodules, reconstituted into liposomes, and assayed for ion channel activity in planar lipid bilayers. These studies demonstrated that nodulin 26 formed a high-conductance ion channel with a maximum unitary conductance of 3.1 ns. The channel exhibited only slight anion-selectivity based on reversal potential measurement. The nodulin 26 ion channel was voltage-sensitive with a tendency to occupy lower conductance states, or to close completely, in the presence of high voltage potentials (>60 mV). Preliminary experiments suggested that phosphorylation may confer voltage sensitivity on the nodulin 26 ion channel. Taken together, these data demonstrate that nodulin 26 can form ion channels in planar lipid bilayers and that vi phosphorylation may be involved in modulating its activity. These observations support a role for nodulin 26 in symbiosome membrane transport and a role for Ca²⁺-dependent phosphorylation in controlling this transport.

Recommended Citation

Weaver, Charles David, "Structural and functional characterization of the nodulin 26 ion channel and the calmodulin-like domain protein kinase from soybean root nodules. " PhD diss., University of Tennessee, 1994.
https://trace.tennessee.edu/utk_graddiss/10608