Doctoral Dissertations

Date of Award

5-2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Daniel M. Roberts

Abstract

Major Intrinsic Proteins (MIPs) are an ancient family of integral membrane proteins that mediate the bidirectional flux of water and small solutes across cellular membranes. Genomic and phylogenetic analyses indicate that plants contain more MIP genes than their animal and microbial counterparts. An analysis of MIP structure also indicates that plant MIPs structurally diverse at the regions that control selectivity of these proteins. Homology modeling was performed using all 35 members of the MIP family from Arabidopsis thaliana. This analysis revealed that MIPs can be divided into 8 functional subgroups based on the amino acids in their selectivity determining ar/R regions. A broader phylogenetic analysis of all available MIP sequences indicates that 92 ar/R regions exist in this dataset, and that much of the diversity arises from plant sources.Homology modeling indicated that the Nodulin 26-like intrinsic protein (NIP) family of Arabidopsis could be divided into two subgroups based on ar/R classification: NIP subgroup I and II. Functional analysis indicates that these two subgroups are functionally distinct. NIP subgroup I forms aquaglyceroporin channels that are also permeable to ammonia, while NIP subgroup II channels are impermeable to water and capable of transporting larger solutes, such as urea. Site-directed mutagenesis studies were used to rationally interconvert the selectivity of these proteins by amino acid substitutions in the ar/R region. Finally, it was demonstrated that members of NIP subgroup II in Arabidopsis form physiologically relevant boric acid channels at the plasma membrane. Nodulin 26, the archetypal NIP, is phosphorylated by a calcium dependant protein kinase (CDPK) at Ser 262 in its C-terminus. This study demonstrates that nodulin 26 phosphorylation increases the water permeability of the channel.In addition, the C-terminus was found to constitute a protein interaction site for nodule cytosolic glutamine synthetase. Finally, the study was extended to characterize MIP channels from the common pea aphid (ApAQP1 and ApAQP2). The results indicate that ApAQP1 is a water-selective aquaporin that is involved in aphid gut osmoregulation, while ApAQP2 is a water channel that is permeable to an array of linear polyols. The potential physiological function of this channel is also discussed.

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