Biomedical subjects

Douglas G Muench

Publications and source records attributed to Douglas G Muench.

8 recordsLinked to original sources

PubMed2006

Biochemical and cellular characterization of the plant ortholog of PYM, a protein that interacts with the exon junction complex core proteins Mago and Y14.

The exon junction complex (EJC) plays an important role in post-transcriptional control of gene expression. Mago nashi (Mago) and Y14 are core EJC proteins that operate as a functional unit in animal cells, and the Mago-Y14 heterodimer interacts with other EJC core and peripheral proteins. Little is known about the biochemical and cellular characteristics of the EJC and its orthologs in plants. Here, we demonstrate that Arabidopsis Mago and Y14 form a ternary complex with PYM, an RNA-binding protein that was previously shown to interact with the Mago-Y14 heterodimer in Drosophila. Fluorescence microscopy indicated that Arabidopsis Mago and Y14 are localized primarily in the nucleus, whereas PYM is mostly cytoplasmic. In vitro pull-down assays using recombinant proteins showed that the amino-terminal region of the Arabidopsis PYM interacts with the Mago-Y14 heterodimer, a similar observation to that previously reported for the animal versions of these proteins. However, we demonstrated also that Arabidopsis PYM has the ability to interact with monomeric Mago and monomeric Y14. Immunoprecipitation and tandem affinity purification from whole cell extracts detected a subtle interaction between the Arabidopsis Mago-Y14 heterodimer and PYM in flowers, indicating that the ternary complex is not abundant in plant cells. The regions of the polypeptide responsible for nuclear import and export were defined using protein truncations and site-directed mutagenesis. This study identifies unique characteristics of Arabidopsis Mago, Y14 and PYM compared to those observed in animal cells. These are predicted to have important functional implications associated with post-transcriptional regulation of gene expression in plant cells.

Amino Acid Sequence↗

PubMed2006

A chloroplast-localized dual-specificity protein phosphatase in Arabidopsis contains a phylogenetically dispersed and ancient carbohydrate-binding domain, which binds the polysaccharide starch.

Dual-specificity protein phosphatases (DSPs) are important regulators of a wide variety of protein kinase signaling cascades in animals, fungi and plants. We previously identified a cluster of putative DSPs in Arabidopsis (including At3g52180 and At3g01510) in which the phosphatase domain is related to that of laforin, the human protein mutated in Lafora epilepsy. In animal and fungal systems, the laforin DSP and the beta-regulatory subunits of AMP-regulated protein kinase (AMPK) and Snf-1 have all been demonstrated to bind to glycogen by a glycogen-binding domain (GBD). We present a bioinformatic analysis which shows that these DSPs from Arabidopsis, together with other related plant DSPs, share with the above animal and fungal proteins a widespread and ancient carbohydrate-binding domain. We demonstrate that DSP At3g52180 binds to purified starch through its predicted carbohydrate-binding region, and that mutation of key conserved residues reduces this binding. Consistent with its ability to bind exogenous starch, DSP At3g52180 was found associated with starch purified from Arabidopsis plants and suspension cells. Immunolocalization experiments revealed a co-localization with chlorophyll, placing DSP At3g52180 in the chloroplast. Gene-expression data from different stages of the light-dark cycle and across a wide variety of tissues show a strong correlation between the pattern displayed by transcripts of the At3g52180 locus and that of genes encoding key starch degradative enzymes. Taken together, these data suggest the hypothesis that plant DSPs could be part of a protein assemblage at the starch granule, where they would be ideally situated to regulate starch metabolism through reversible phosphorylation events.

Amino Acid Sequence↗

PubMed2005

The PII signal transduction protein of Arabidopsis thaliana forms an arginine-regulated complex with plastid N-acetyl glutamate kinase.

The PII proteins are key mediators of the cellular response to carbon and nitrogen status and are found in all domains of life. In eukaryotes, PII has only been identified in red algae and plants, and in these organisms, PII localizes to the plastid. PII proteins perform their role by assessing cellular carbon, nitrogen, and energy status and conferring this information to other proteins through protein-protein interaction. We have used affinity chromatography and mass spectrometry to identify the PII-binding proteins of Arabidopsis thaliana. The major PII-interacting protein is the chloroplast-localized enzyme N-acetyl glutamate kinase, which catalyzes the key regulatory step in the pathway to arginine biosynthesis. The interaction of PII with N-acetyl glutamate kinase was confirmed through pull-down, gel filtration, and isothermal titration calorimetry experiments, and binding was shown to be enhanced in the presence of the downstream product, arginine. Enzyme kinetic analysis showed that PII increases N-acetyl glutamate kinase activity slightly, but the primary function of binding is to relieve inhibition of enzyme activity by the pathway product, arginine. Knowing the identity of PII-binding proteins across a spectrum of photosynthetic and non-photosynthetic organisms provides a framework for a more complete understanding of the function of this highly conserved signaling protein.

Arabidopsis↗

PubMed2005

The peroxisomal multifunctional protein interacts with cortical microtubules in plant cells.

BACKGROUND: The plant peroxisomal multifunctional protein (MFP) possesses up to four enzymatic activities that are involved in catalyzing different reactions of fatty acid beta-oxidation in the peroxisome matrix. In addition to these peroxisomal activities, in vitro assays revealed that rice MFP possesses microtubule- and RNA-binding activities suggesting that this protein also has important functions in the cytosol. RESULTS: We demonstrate that MFP is an authentic microtubule-binding protein, as it localized to the cortical microtubule array in vivo, in addition to its expected targeting to the peroxisome matrix. MFP does not, however, interact with the three mitotic microtubule arrays. Microtubule co-sedimentation assays of truncated versions of MFP revealed that multiple microtubule-binding domains are present on the MFP polypeptide. This indicates that these regions function together to achieve high-affinity binding of the full-length protein. Real-time imaging of a transiently expressed green fluorescent protein-MFP chimera in living plant cells illustrated that a dynamic, spatial interaction exits between peroxisomes and cortical microtubules as peroxisomes move along actin filaments or oscillate at fixed locations. CONCLUSION: Plant MFP is associated with the cortical microtubule array, in addition to its expected localization in the peroxisome. This observation, coupled with apparent interactions that frequently occur between microtubules and peroxisomes in the cell cortex, supports the hypothesis that MFP is concentrated on microtubules in order to facilitate the regulated import of MFP into peroxisomes.

Binding Sites↗

PubMed2004

Large-scale identification of tubulin-binding proteins provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells.

Microtubules play an essential role in the growth and development of plants and are known to be involved in regulating many cellular processes ranging from translation to signaling. In this article, we describe the proteomic characterization of Arabidopsis tubulin-binding proteins that were purified using tubulin affinity chromatography. Microtubule co-sedimentation assays indicated that most, if not all, of the proteins in the tubulin-binding protein fraction possessed microtubule-binding activity. Two-dimensional gel electrophoresis of the tubulin-binding protein fraction was performed, and 86 protein spots were excised and analyzed for protein identification. A total of 122 proteins were identified with high confidence using LC-MS/MS. These proteins were grouped into six categories based on their predicted functions: microtubule-associated proteins, translation factors, RNA-binding proteins, signaling proteins, metabolic enzymes, and proteins with other functions. Almost one-half of the proteins identified in this fraction were related to proteins that have previously been reported to interact with microtubules. This study represents the first large-scale proteomic identification of eukaryotic cytoskeleton-binding proteins, and provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells.

Arabidopsis↗

PubMed2004

Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production?

Plant scientists have long recognized the need to develop crops that absorb and use nutrients more efficiently. Two approaches have been used to increase nutrient use efficiency (NUE) in crop plants. The first involves both traditional breeding and marker-assisted selection in an attempt to identify the genes involved. The second uses novel gene constructs designed to improve specific aspects of NUE. Here, we discuss some recent developments in the genetic manipulation of NUE in crop plants and argue that an improved understanding of the transition between nitrogen assimilation and nitrogen recycling will be important in applying this technology to increasing crop yields. Moreover, we emphasize the need to combine genetic and transgenic approaches to make significant improvements in NUE.

Agriculture↗

PubMed2002

Expression and purification of the chloroplast putative nitrogen sensor, PII, of Arabidopsis thaliana.

The bacterial PII protein was discovered over 30 years ago and is known to be a key player in orchestrating the coordination of nitrogen metabolism with changes in carbon flux. Bacterial PII is regulated by covalent modification and binding to effector molecules in response to the nitrogen/carbon status of the cell and appropriately coordinates the activity of glutamine synthetase and the transcription of a nitrogen sensitive regulon. Recently, a PII protein was identified in higher plants and the protein was found to be localized to the chloroplast. The Arabidopsis thaliana putative nitrogen sensor protein, PII, was cloned and overexpressed with a C-terminal 6-histidine tag. The full-length protein, which included the chloroplast transit peptide, was overexpressed in Escherichia coli, but was very susceptible to proteolytic degradation. Removal of the transit peptide yielded a highly pure, stable recombinant protein whose identity was established as PII by matrix assisted laser desorption ionization-time of flight mass spectrometry. Polyclonal antibodies generated against the recombinant protein effectively immunoprecipitated PII from an A. thaliana extract and the protein was confirmed to be 17 kDa in mass. The availability of milligram amounts of PII will allow a complete biophysical characterization of the protein and antibodies should aid in the identification of PII interacting proteins and the establishment of the higher plant PII signal transduction cascade.

Amino Acid Sequence↗

PubMed2001

Identification of a rice RNA- and microtubule-binding protein as the multifunctional protein, a peroxisomal enzyme involved in the beta -oxidation of fatty acids.

The control of subcellular mRNA localization and translation is often mediated by protein factors that are directly or indirectly associated with the cytoskeleton. We report the identification and characterization of a rice seed protein that possesses both RNA and microtubule binding activities. In vitro UV cross-linking assays indicated that this protein binds to all mRNA sequences tested, although there was evidence for preferential binding to RNAs that contained A-C nucleotide sequence motifs. The protein was purified to homogeneity using a two-step procedure, and amino acid sequencing identified it as the multifunctional protein (MFP), a peroxisomal enzyme known to possess a number of activities involved in the beta-oxidation of fatty acids. The recombinant version of this rice MFP binds to RNA in UV cross-linking and gel mobility shift experiments, co-sediments specifically with microtubules, and possesses at least two enzymatic activities involved in peroxisomal fatty acid beta-oxidation. Taken together these data suggest that MFP has an important role in mRNA physiology in the cytoplasm, perhaps in regulating the localization or translation of mRNAs through an interaction with microtubules, in addition to its peroxisomal function.

Amino Acid Sequence↗