Masters Theses

Date of Award

12-2000

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Daniel M. Roberts

Committee Members

Elizabth Howell, Jim Hall

Abstract

Bradyrhizobium japonicum bacteria infect the roots of soybeans resulting in the formation of a symbiotic, nitrogen-fixing nodule. Within the nodule, the bacteria are enclosed in a specialized organelle called the symbiosome. The bacteria are separated from the plant cell cytosol by the symbiosome membrane (SM). The formation of nodules is marked by the expression of several nodule-specific proteins called nodulins.In soybean, the nodulin 26 protein constitutes at least 10% of the total membrane protein and has been identified as a member of the major intrinsic protein (MIP) family.

Nodulin 26 is a multifunctional aquaglyceroporin that allows the flux of both water and solutes. Besides its multifunctional transport properties, nodulin 26 has a low intrinsic water transport rate that is 50-fold lower than that of aquaporin 1, the"prototypical" water-transport aquaporin of the MIP family. To determine the factors that may contribute to the multifunctional transport properties of nodulin 26 and its regulation, we used the heterologous Xenopus expression system to investigate residues important for the low intrinsic transport property of nodulin 26, we tested the functional roles of AQPl-hke mutations of nodulin 26: the glutamine at position 114 of nodulin 26 was substituted by an arginine (Q114R), the cysteine at position 172 was substituted by a histidine (C172F), the valine at position 197 was substituted by a histidine (V197H), and the isoleucine at position 226 was substituted by a histidine (I226H). Findings indicate that two mutants (I226H and Q114R) are expressed and form water channels. Based on comparisons of water and glycerol permeabilities, Q114R is identical to nodulin 26 suggesting that this substitution does not confer higher water permeability or selectivity in aquaporin 1. I226H exhibits a lower permeability even though its expression appears to be normal. The other mutations (C172F and V197H) result in dysfunctional expression and/or targeting

Comparison of the sequences of glycerol and water-selective aquaporins indicate that there are five "discriminant" residues that are invariant within each of these groups.Notable, the nodulin 26 sequence is a hybrid of the consensus sequences for aquaporins and glycerol facilitators. Weinvestigated two of these "discriminant" residues in nodulin26 to see if they affected its water and glycerol transport properties. The leucine at position 230 was mutated to a tryptophan (AQP-like) (L230W) and the tyrosine at position 229 was mutated to a proline (glycerol transporter-like) (Y229P). Water and glycerol permeability assays revealed that both L230W and Y229P were no longer fluxing water or glycerol above the water-injected control oocytes, again because of defects in targeting. The difficulties with improper expression, folding or trafficking to the plasma membrane made it difficult for us to draw any conclusions about the influences of these residues in the selectivity or single channel rate of nodulin 26.

The low single channel rate observed for nodulin 26 suggested that nodulin 26 may undergo regulation via external signals Analysis of oocytes expressing nodulin 26 show that the relatively low water permeability of nodulin 26 is enhanced by reducing thepH. The highest water permeability was observed at pH 5.5 which was increased 3-fold compared to that observed at the standard pH of 7.6. Conversely, calcium was found to be a negative regulator of nodulin 26. A decrease in extracellular calcium increased the water permeability at least 2-fold. Conversely, an increase in external calcium exerted the opposite effect, with the relative permeability being reduced by 2-fold. Use of the calcium chelator, ethylbis(ocyethylene-nitilo)tetraacetic acid, in the recording bath further elevated the water permeability by 3-fold. The microinjection of another calcium chelator, l,2-bis(2aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, elevated the water permeability by 4-fold suggesting that the calcium sensor is internal.

Previous studies indicate that nodulin 26 is the major phosphoprotein on the SM. The phosphorylation occurs at serine 262 of nodulin 26 and is catalyzed by a calcium-dependent SM-associated protein kinase of the calcium-dependent protein kinase(CDPK)family. To determine the functional effect of phosphorylation, the effects of a constitutive recombinant CDPK on the ability of nodulin 26 to transport water and glycerol using the Xenopus laevis oocytes expression system were determined. Oocytes injected with nodulin 26 alone showed an enhanced rate of oocyte swelling, whereas nodulin 26 oocytes injected with CDPK showed a 2-fold reduction in Pf. To test whether the result is specific for serine 262, a nodulin 26 mutant (serine to alanine at position 262) was generated that was unable to be phosphorylated at the 262 position. In contrast to wild-type nodulin 26, the water permeability of the alanine mutant was not affected by CDPK injection. In addition, glycerol transport properties were also made to determine if phosphorylation by CDPK affects the glycerol permeability of nodulin 26. Similar to the results obtained with the water permeability, nodulin 26 oocytes injected with CDPK showed a significant reduction in glycerol permeability compared to the nodulin 26 oocytes not injected with CDPK. Again, the alanine mutant did not show any significant reduction in glycerol permeability upon CDPK injection. Western blot analysis revealed that the decrease in nodulin 26 observed in CDPK-injected oocytes appears to be the result of a reduced level of nodulin 26 on the plasma membrane, raising the possibility that phosphorylation of nodulin 26 affected membrane trafficking.

To assay the short-term effects of phosphorylation on the intrinsic transport rate of nodulin 26, the effects of phosphorylation of nodulin 26 by the endogenous Xenopus protein kinase C was determined. A synthetic peptide corresponding to the carboxyl terminus of nodulin 26, CK-15, was shown to be a protein kinase C substrate with a Kmof520 pM,suggesting that nodulin 26 is a protein kinase C substrate. To test the effect of phosphorylation on nodulin 26 activity, we used a protein kinase C agonist (phorbol-12-myristate-13-acetate, TPA) and a protein phosphatase inhibitor (okadaic acid). Both agents were shown to stimulate the water permeability of nodulin 26 by 3-fold. In contrast, mutants of nodulin 26 that do not possess a phosphorylatable residue at position 262 were not affected by either okadaic acid or TPA.

Overall the data show that the water transport property of nodulin 26 was enhanced under conditions of low pH, low intracellular calcium levels, or phosphorylation. The regulation of nodulin 26 by pH, calcium, and phosphorylation may contribute to the regulation of nodulin 26 in the SM The regulation of nodulin 26 by these factors would permit the protein to shift between an activated, higher permeability state to a less active, lower permeability state to allow osmoregulation and possibly adaptation to environmental factors

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