Doctoral Dissertations

Date of Award

5-2002

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Daniel M. Roberts

Committee Members

John Dunlap, Wesley Wicks, Albrect Von Armin, Jim Hall

Abstract

By isolating RNA from mature nodules of the legume Lotus japonicas and employing an RT-PCR approach, two new cDNAs were characterized whose open reading frames code for members of the MIP (Major Intrinsic Protein) family of membrane proteins. These two genes products were termed (Lotus Intrinsic Membrane Protein 1 and 2) LIMP1 and LIMP2. Both LIMP1 and LIMP2 display all the hallmarks of the MIP protein family including, six putative transmembrane domains based on hydropathy plots, and the invariant NPA (asparagine-proline-alanine) signature motifs located symmetrically on two loops, which connect transmembrane helix 3-4 and transmembrane helix 5-6. Based on sequence information, LIMP1 is placed in the TIP (Tonoplast membrane Intrinsic Protein) subfamily of plant MIP proteins. Based on localization within the cells, TIPs are typically found on the tonoplast membrane of plant vacuoles. Functional characterization of LIMP1 by expression in Xenopus oocytes has demonstrated that it is a waterselective aquaporin. Using Northern blot techniques combined with immunolocalization, the expression patterns for LIMP1 were analyzed. The results indicate that LIMP1 is expressed predominately in nodules and roots. Based on Western blot analysis, however the highest LIMP1 protein levels are found in roots. LIMP1 appears to be regulated by diurnal cycling conditions, with expression increasing during the day hours, but falling off at the beginning of the night cycle. Increases in LIMP1 expression precedes the time of the diurnal cycle when the plant rate is the highest. This finding suggests that LIMP1, similar to other aquaporins, is regulated in response to the need for trancellular water uptake and transport from the soil during times of high transpiration stream activity.

Although the LIMP2 protein shows some sequence similarity to LIMP1, it displays several features which distinguish it. Based on primary sequence identities, LIMP2 is classified in the plant NIP (Nodulin-like Intrinsic Protein) subfamily of MIP proteins which takes its name from the patriarch protein, nodulin 26. Nodulin 26 is a soybean nodule protein localized exclusively to the symbiosome membrane which surrounds the symbiotic bacteria during the nitrogen fixation process. Comparison of the LIMP2 protein sequence with other MIP family members shows that it shares the highest sequence identity (68%) with nodulin 26. In addition to structural similarity, nodulin 26 and LIMP2 also share the ability to facilitate the transport of water as well as small uncharged solutes (e.g. glycerol) upon expression in Xenopus oocytes. Analysis of the tissue distribution of LIMP2 by Northern blot and immunolocalization reveal that the LIMP2 transcript is expressed only in nodule tissue and that this protein, like nodulin 26, is present on the symbiosome membrane.

LIMP2 and LIMP1 both show the ability to transport water, but the rate of transport is several-fold lower than that observed for high water transport MIP proteins such as mammalian aquaporin 1 (AQP1). Based on the crystal structures of AQP1 and the bacterial glycerol facilitator protein (GlpF), models of the selectivity filter for LIMP1 and LIMP2 were generated and analyzed in order to resolve the underlying characteristics for these observed differences. The proposed selectivity filter for LIMP2 displays a pore large enough to accommodate the passage of glycerol. Further, three key residues, a tryptophan in helix 2 and a valine in helix 5 and an arginine in the second NPA loop, form an amphipathic signature similar to the selectivity filter of the glyceroporin GlpF and are proposed to account for the glycerol permeability of nodulin 26 and LIMP2. In silica modeling of the predicted pore for LIMP1/TIPs shows a unique set of features. It shows a higher hyrdrophobic character than AQP1 and unusual substitutions, a histidine in helix 2 which replaces an invariant phenylalanine, an isoleucine in helix 5 which substitutes for a histidine, and a valine in the second NPA loop which substitutes for an invariant arginine. These substitutions suggest that the pore structure of LIMP1 is quite unlike the two models (AQP1 and GlpF), and further insight into factors that contribute to its rate and selectivity will have to await more detailed structural analysis.

In addition to sequence and functional similarities, both nodulin 26 and LIMP2 share a conserved phosphorylation motif for CDPK (Calcium Dependent Protein Kinase) located on the cytosolic carboxyl terminal tail. In vitro assays using the carboxyl terminal sequence of LIMP2 suggest that similar to nodulin 26, it is phosphorylated by CDPK and is likely a target for calcium-dependent phosphorylation. Using an antibody which specifically recognizes the phosphorylated form of nodulin 26, a developmental pattern of phosphorylation is observed, where immature nodules show no or very low levels of phosphorylation and very old nodules show no phosphorylation of nodulin 26 although the protein is present in both young and old nodules at the same level as in mature nodules. Using this phosphorylation-specific antibody it was also determined that in mature soybean nodules the inherent level of phosphorylation is stimulated in response to osmotic stress (drought and salinity).

Taken together, these results show that Lotus japonicus nodules have two MIP proteins. LIMP1 is a member of the TIP family of plant MIP proteins which is found predominately in root tissues where it forms a water selective channel, and its expression is controlled by diurnal variation. Its role in roots and nodules remains unknown, but it could play a role in transcellular water flow and cell volume regulation as proposed for other TIPs. In contrast, LIMP2 appears to be the Lotus japonicus ortholog of nodulin 26, an aquaglyceroporin that is localized on the symbiosome. LIMP2 and nodulin 26 are phosphorylated and likely regulated by calcium signaling through a symbiosome membrane CDPK. Previous studies show that phosphorylation of nodulin 26 enhances its water permeability. The present work suggests that phosphorylation by a calcium-dependent kinase is stress regulated and that phosphorylation of nodulin 26 may be among the osmoregulatorry responses of nodules necessary for stress adaptation, possibly by facilitating rapid water/solute flow to buffer the infected cell cytosol and symbiosome to fluctuations in osmotic gradients.

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