Dring N. Crowell - US grants

Compounds derived from mevalonic acid are required for growth of plant and animal cells. In plants, the mevalonic acid pathway participates in the synthesis of cytokinins, abscisic acid, gibberellins, ubiquinones, plastoquinones, dolichols, sterols, carotenoids, chlorophylls, and numerous other isoprenoids. However, it is not clear whether these small isoprenoids fully account for the mevalonic acid growth requirement of plant cells. In mammalian and yeast cells, it has been shown that products of mevalonic acid are covalently bound to G-proteins, low molecular weight ras-like GTP-binding proteins and nuclear lamins. Covalent attachment of isoprenoids to these proteins is required for their assembly into membranes and, hence, for their role in signal transduction and cell growth control. Given the recent discovery of G-proteins and ras-like GTP-binding proteins in plants, it was predicted that plants also contain isoprenylated proteins. Preliminary data shown in this proposal indicate that plants contain isoprenylated, membrane-associated proteins similar in mass to ras-like GTP-binding proteins and nuclear lamins. The goals of this project are to determine the structure of the isoprenoid modification of these proteins and to identify the proteins. This work will lead to and understanding of the role of protein isoprenylation in plant cell growth, phytohormone action, signal transduction, and membrane biogenesis. %%% Isoprenoids belong to a specialized class of lipid molecules. In animal cells, many proteins have been shown to be linked to isoprenoids. These proteins participate in cell division and other cellular processes. Current evidence suggests that the isoprenoid modification is essential for the biological activity of these proteins. This proposal presents the first evidence that plant cells also contain isoprenylated proteins. The goals of this project are to determine the chemical nature of the isoprenoids that are linked to plant proteins and to identify the respective proteins. This work will lead to an understanding of how the isoprenoid modification of proteins affects plant growth and development.

9877094

Abstract

This project involves the acquisition of a phosphoimager system for molecular biology research and training. The instrument will be housed in the Department of Biology at Indiana University-Purdue University at Indianapolis and will serve 5 major and two minor users in the department of Biology and two major users in the Department of Chemistry. All major and minor users employ conventional autoradiography, fluorography and/or chemiluminescent detection with film on a routine basis. However, these techniques are limited by the narrow linear response range of film and by the lengthy exposure times required for the fluorographic detection of weak energy isotopes such as 3H and 14C. Phosphoimager analysis has several key advantages over autoradiography and fluorography. First, phosphoimager screens have a linear response range of five orders of magnitude as opposed to only two for autoradiography. Second, phosphoimager exposure times are generally only one tenth those required for autoradiography and fluorography. This is especially advantageous for the detection of weak energy isotopes where fluorography exposure times of weeks or months are often required. Third, the phosphoimager data can be quantified and imported into computer drawing programs for accurate quantitative analysis. This system will complement existing molecular biology equipment in the laboratories of the major and minor users. Moreover, the high degree of accuracy and efficiency afforded by phosphoimager analysis will improve the quality and quantity of molecular biology research and teaching performed by the investigators.

Sen

Juvenile hormone is the common name given to one or more structurally related compounds that are produced by insects and are essential for regulating insect growth and development. The primary objective of this proposal is to better understand how moths make homologous juvenile hormone and in so doing, gain insight for how insect metabolism differs from other animals. Moths are unique with regards to juvenile hormone metabolism- in this insect, the hormone is a heterogeneous mixture of compounds that differ structurally from one another by the presence of one to three extra carbons. Moth juvenile hormone levels appear to be highly regulated, although the functional role of each of the homologues is not understood.

The specific aim of the research is to isolate and characterize the enzyme, prenyltransferase, which catalyzes the condensation of isoprene units (or in the case of moths, homologous isoprene units) and is a required event in juvenile hormone production. Prenyltransferase will first be isolated from the endocrine gland that synthesizes the hormone, using both conventional protein purification methods and recombinant DNA technology. These initial experiments will focus on ascertaining how many and what kind of prenyltransferase is present in moths and which of these are responsible for juvenile hormone production. Subsequently, we will analyze the structural differences between juvenile hormone producing and other prenyltransferases, by evaluating their variability in amino acid sequence, catalytic properties, 3D-structure (to be determined computationally), and expression profiles. For the latter, we will specifically examine whether juvenile hormone producing prenyltransferase is developmentally regulated and whether its levels (both transcriptional and translational) parallel those of hormone production, indicating the role of prenyltransferase in regulating juvenile hormone production (and structure) in moths.

The goal of this project is to gain a better understanding of the biosynthesis, compartmentation,and function of plant isoprenoids. Recent work in several laboratories has revealed the presence of two distinct isoprenoid biosynthetic pathways in plants: a mevalonate-dependent pathway that is responsible for the synthesis of sterols, brassinosteroids, cytokinins and other terpenoids, and a mevalonate-independent pathway that is involved in the production of plastidic isoprenoids (plastoquinones, phytyl tail of chlorophylls, carotenoids, etc.). However, much remains to be learned of the interactions between these different isoprenoid compartments in plant growth and development. To address this complex problem, this PI has identified mutants of Arabidopsis thaliana that are resistant to lovastatin (lvr ), a specific inhibitor of mevalonate synthesis that severely impairs root growth of wild type seedlings.One of these mutants, lvr 111, exhibits extreme resistance to lovastatin and is defective in chlorophyll and carotenoid pigmentation. Interestingly, F2 population analysis suggests that both phenotypes are caused by a single mutation. Since this mutant exhibits alterations in the cytoplasmic pathway (lovastatin resistance) as well as the plastidic pathway (pigmentation defect), the PI will test the hypothesis that the communication between these isoprenoid compartments is altered in the lvr 111 mutant. This will be accomplished by positional cloning and characterization of the LVR 111 gene, which will provide insights into the flux and/or function of isoprenoid compounds within plant cells.

In plant and animal cells, proteins must be delivered to appropriate sites of action and often must interact with other proteins for normal function. Protein prenylation, the attachment of an isoprenoid lipid to a protein, is one mechanism by which proteins are targeted to appropriate sites and/or modified for interaction with other proteins. While this process has been studied in detail, the degradation of prenylated proteins and the fate of the isoprenoid lipid have not been described in plants. The degradation of prenylated proteins results in the release of a prenylcysteine (i.e., either farnesylcysteine or geranylgeranylcysteine) with the isoprenoid attached to the amino acid cysteine via thioether linkage. Prenylcysteine metabolism in plants is important, at least in part, because prenylcysteine compounds cause an enhanced response to the plant hormone abscisic acid (ABA). This project will examine the farnesylcysteine lyase enzyme that metabolizes farnesylcysteine to farnesal and cysteine in the cruciferous plant Arabidopsis thaliana. A gene on chromosome 5 of the Arabidopsis genome encodes this enzyme and mutations in this gene cause an enhanced response to ABA, presumably because of farnesylcysteine accumulation. This project will also examine the metabolism of geranylgeranylcysteine, which proceeds by a different mechanism. Thus, plants possess at least two distinct mechanisms for prenylcysteine metabolism, one for farnesylcysteine and one for geranylgeranylcysteine, and these processes are required for normal ABA responsiveness. To test the hypothesis that the enzymes involved in prenylcysteine metabolism are important for isoprenoid salvage and detoxification and to describe in detail the role of prenylcysteine metabolism in ABA signaling, biochemical analyses on Arabidopsis prenylcysteine metabolic enzymes will be performed and the corresponding genes identified. In addition, the subcellular locations of Arabidopsis prenylcysteine metabolic enzymes and the phenotypes of Arabidopsis plants deficient in prenylcysteine metabolic enzymes will be analyzed. Finally, the regulation of genes encoding prenylcysteine metabolic enzymes in Arabidopsis will be examined. This work will significantly contribute to a better understanding of the metabolism of prenylated proteins and the role of prenylcysteine salvage/detoxification in plant growth and development.

Broader Impacts
The project will provide an excellent training environment for undergraduate and graduate students. IUPUI students will be recruited to work on this project from the Diversity Scholars Research Program, which is designed to encourage undergraduate women and minorities interested in graduate school to pursue undergraduate research opportunities, and the McNair Scholars Program, a federally funded program to increase the number of low-income, first-generation, underrepresented students in Ph.D. degree programs. Given the central location and urban environment of the IUPUI campus, it is expected that more young scientists, including women and minorities, will receive professional training through participation in this project.