Identification of a Rice RNA- and Microtubule-binding Protein as the Multifunctional Protein, a Peroxisomal Enzyme Involved in the β-Oxidation of Fatty Acids

Abstract

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 ␤-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 ␤-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.

Large-scale Identification of Tubulin-binding Proteins Provides Insight on Subcellular Trafficking, Metabolic Channeling, and Signaling in Plant Cells

“…Two studies demonstrated that the SDS-PAGE profiles of plant proteins that eluted from tubulin and MT affinity chromatography columns were qualitatively similar (5,13), and essentially all proteins that bound to the tubulin affinity columns subsequently co-sedimented with MTs in pelleting assays (14). This indicates that tubulin affinity chromatography can be used to successfully purify MT-binding proteins.…”

“…These proteins, often called MT-interacting proteins (MIPs) (2,4), likely bind to MTs as a mechanism to regulate their own activity, to direct their subcellular localization, or as a concentrating mechanism at specific locations within the cell. The interaction of these proteins with MTs is often transient, making it difficult to visualize their interaction with MTs in situ (5). The large surface area provided by the MT network, and the cytoskeleton as a whole, likely serves as a matrix for the binding of hundreds of proteins to an extent that is dependent on cell type and environmental conditions (6).…”

“…This indicates that tubulin affinity chromatography can be used to successfully purify MT-binding proteins. Indeed, tubulin affinity chromatography has been used to identify three authentic plant MT-binding proteinselongation factor-1␣ (EF-1␣) (15), the peroxisomal multifunctional protein (5), and phospholipase D (14)-as well as additional authentic and putative MT-binding proteins from both plant and animal cells (16 -18).…”

ArabidopsisPEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A Cooperate in Cell Cycle–Associated Replication of Peroxisomes

“…Because each half of the YFP is not intrinsically fluorescent, YFP fluorescence is observed only when intermolecular interactions occur between nYFP-and cYFPtagged proteins (Citovsky et al, 2006). All cells were also cobombarded with a plasmid encoding a red fluorescent protein linked to the rice multifunctional protein (RFP-MFP), a wellcharacterized peroxisomal matrix enzyme (Chuong et al, 2002(Chuong et al, , 2005. Coexpressed RFP-MFP served as an internal standard for transformation efficiency as well as a convenient peroxisomal marker to assess colocalizations with BiFC signals and potential changes in peroxisome morphology.…”

Función del gen 3-cetoacil-CoA tiolasa 2 (KAT2) en defensa y desarrollo. Participación de los peroxisomas en las respuestas a herida en Arabidopsis thaliana.

“…En otras especies vegetales como pepino, también se han caracterizado distintas isoenzimas MFP expresadas diferencialmente en distintos tejidos de la planta y con distintas actividades en cada caso (Behrends et al, 1988;Preisig-Muller et al, 1994; Guhnemann-Schafer y Kindl, 1995a;. Por último, las proteínas MFP presentan varias regiones de unión a microtúbulos que le permiten el transporte a través de los mismos, e implica que estas proteínas se encuentren asociadas a microtúbulos además de su localización peroxisomal (Chuong et al, 2002; Kindl, 1993), mango (Bojorquez y Gómez-Lim, 1995), colza (Olesen y Brandt, 1995), cotiledones de calabaza (Kato et al, 1996), girasol (Oeljeklaus et al, 2002) y en Arabidopsis (Hayashi et al, 1998b;Germain et al, 2001). El genoma de Arabidopsis contiene tres genes que codifican proteínas KAT.…”