Richard N. Trelease - US grants

The major goal of this research proposal is to generate new information on the biogenesis of a plant peroxisome, the glyoxysome, which is pivotally involved in storage lipid metabolism during the critical heterotrophic growth phase of economically - important oilseeds. The main emphasis is on deciphering the mechanisms responsible for targeting and importing membrane phospholipids and enzyme proteins into glyoxysomes in maturing and germinated cotton seeds. Understanding organelle biogenesis (integral to intracellular compartmentation) is an essential part of ultimately comprehending the complexities of cellular metabolism. Formation of glyoxysomes will be analyzed by electron microscopy coupled with immunocytochemistry of enzymes to visualize images in thin sections of developing cotyledons. Phospholipid composition of organelle membranes will be quantitated by Iatrascan TLC/FID, and the subcellular site of phospholipid synthesizing enzymes will be determined from radioactive labeling assays of fractions prepared by density - gradient centrifugation. In vivo (protoplasts) and in vitro (translation products from Poly A+ mRNA) systems will be established to experimentally evaluate critical requirements for import of isocitrate lyase, catalase, and malate synthase into peroxisomes isolated from cotton and other species. Recombinant DNA experiments will be initiated wherein a lambda gt10 library will be sequenced and those with full length open reading frames will be subcloned into vectors for establishing an in vitro transcription/translation system designed to assess molecular modifications on the import of proteins. The timing and appearance of gene transcripts in maturing seeds will be determined with cloned DNA probes to document apparent translation control(s) of enzyme synthesis in maturing seeds.***

Three different aspects of plant peroxisome biogenesis will be pursued: (1), the means by which the expanding membrane of oilseed glyoxysomes acquires its protein and lipid is an integral part of the organelle biogenesis. The hypothesis will be tested that lipid bodies contribute membrane lipid to differentiating oilseed glyoxysomes; (2), parameters and conditions necessary and/or sufficient for recognition and tranlocation in vivo of malate synthase, isocitrate lyase and catalase into peroxisomes of cultured monkey kidney and selected plant cells will be determined; (3) studies will continue on deciphering patterns of regulation and developmental expression of catalase isoforms during the changeover from glyoxysomes to leaf-type peroxisomes, and of isocitrate lyase and malate synthase during glyoxysome differentiation in maturing and germinated seeds. The availability of an embryo culture system is expected to provide a unique means to help resolve regulation patterns within maturing seeds. Peroxisomes occur in virtually all eukaryotic cells, conducting a variety of essential metabolic functions depending on the cell type in which they reside. They are not capable of synthesizing their own phospholipids or proteins, and are therefore dependent upon the selective and complicated intracellular trafficking mechanisms responsible for their biogenesis. The objectives of this proposal are to examine the molecular and biochemical bases of selected, interrelated aspects of peroxisome biogenesis; the emphasis will be on cottonseed glyoxysomes.

Three specific objectives are proposed. The first is to develop an in-vivo plant cell import system for experimentally determining necessary and sufficient C-terminal targeting signals on plant peroxisome proteins. Preliminary data indicate that the GUS gene with an appended S-K-L-COOH and delivered biolistically to cultured tobacco cells, is directed to putative peroxisomes. Histochemical and double indirect immunofluorescent staining suggest this system will be worth pursuing. The second objective is to discover whether the C-terminal tripeptides on plant peroxisome proteins are necessary and/or sufficient for import in vivo into peroxisomes of cultured mammalian cells. In preliminary studies, the C-terminal tripeptide A-R-M of cottonseed isocitrate lyase was found necessary for import into CV-1 monkey kidney and CHO cells. The third objective is to discover the targeting signal(s) necessary and sufficient for import of catalases into peroxisomes of plant and mammalian cells. There are essentially no data elucidating the specific putative targeting signal(s) for any eukaryotic catalase, the basic constitutive enzyme of all peroxisomes. Through unexpected preliminary results, a means was discovered whereby it is possible to distinguish experimentally-imported, native catalase from endogenous peroxisomal catalase. This breakthrough will permit the determination of whether internal signal(s) within C- or N-terminal domains direct catalase to eukaryotic peroxisomes. %%% Compartmentation of enzyme reactions and/or entire metabolic pathways within organelles is essential to the overall coordination of cellular processes and functions of all eukaryotic cells. Understanding biogenesis, differentiation, and function(s) of each organelle is essential before we can comprehend the complex, integrated functions of cells. One of the most active areas of cell biological research which pertains directly to cell regulation and function is intracellular protein trafficking to organelles. The overall goal of the proposed research is to discover and elucidate molecular targeting signals on enzymes which are directed to, and translocated into, plant cell peroxisomes. Peroxisomes occur in virtually all eukaryotic cells and conduct a variety of essential metabolic function(s) related to the cell type in which the peroxisome resides. Recent experimental research on peroxisome targeting signals revealed that a C-terminal tripeptide motif was necessary and sufficient for import into mammalian and yeast peroxisomes. Surveys of published DNA sequences and western blot screens with antisera to a "consensus" tripeptide (S-K-L) indicated that the motif was common for most, but clearly not all, peroxisome enzymes among a variety of eukaryotic organisms. Evidence also exists for some internal and N-terminal cleavable peroxisome signals. A primary reason that experimental data do not exist for targeting signals on any native plant peroxisome enzyme is that import systems, either in vitro or in vivo, have not been developed for plant cells. The aim of this project is to close that informational gap by developing and exploiting an experimentally manipulable in vivo plant peroxisomal import assay system.

DNA sequence determinations are of fundamental importance for studies in many areas of the biosciences. including functional biochemistry, cell and molecular biology, population genetics, and molecular systematics. The purpose of this proposal is to streamline DNA sequencing at Arizona State University by seeking funds to establish a DNA sequencing facility. The major piece of equipment for this facility will be an automated DNA sequencer, which currently is not available at our University and which is requested in this proposal. About 20 groups at Arizona State University routinely use DNA sequencing in their research, and a large number of graduate students, undergraduates, postdoctoral fellows, staff, and faculty will benefit from the establishment of a DNA sequencing facility with an automated sequencer. The sequencer requested utilizes an infrared laser diode to excite fluorophor-labeled DNA that is size-separated by gel electrophoresis. Fluorescence from the fluorophor is detected, and simultaneous scanning of four sequencing lanes over time can provide a DNA sequence of 800 nucleotides or more with over 99% accuracy in a highly cost-effective manner. With the equipment requested, 22 samples can be sequenced simultaneously. Apart from providing a 30% cost-share in purchasing this equipment, the University also will create a staff-level position for an individual to run the DNA sequencing facility and will bear most of the personnel cost involved. A Ph.D.-level research scientist (already on staff) will supervise the facility. Nominal user fees (about $5 per sequence) will be charged to cover the cost of chemicals, the DNA sequencer service contract, and a small part (about 20%) of the technician's salary. This facility will provide a highly effective and economic mechanism to obtain and analyze DNA sequence information. An additional benefit is that DNA sequencing becomes accessible to a large number of graduate students who work in research areas (for examp le, systematics and population biology) that would benefit from DNA analysis, but for whom DNA sequencing facilities currently are not readily available. Also, many undergraduate projects, which usually preclude use of radioactivity, could include DNA sequencing. Another significant advantage of acquisition of an automated DNA sequencer is that when DNA sequencing becomes more efficient, students will be able to gather and analyze sequence information more expediently, leaving more room for emphasis on training in other important areas.

9728935 Trelease Eukaryotic cells contain membrane-enclosed compartments (organelles) in which various cellular functions are carried out. This isolation of various functions within physical boundaries is critical to the life of the cell: it allows the cell to regulate the functions of each compartment separately; and it prevents the contents of each compartment from interfering with others. However, it does require that the cells have mechanisms for sorting and translocating individual proteins to their proper subcellular locations. One type of intracellular compartment is the peroxisome, which occurs in virtually all eukaryotic cells. Peroxisomes participate in a very wide range of metabolic pathways, depending on the cell or tissue type in which they function. The overall goal of this project is to understand the complicated means by which peroxisomal membrane proteins (PMPs) become selectively integrated into the boundary membrane of peroxisomes (glyoxysomes) in oilseed seedlings, and to understand the functions of such PMPs within the membrane as the seedlings develop. Three specific objectives are proposed relative to the biogenesis of plant peroxisomes. The first aim is to discover the putative membrane peroxisomal targeting signal (mPTS) responsible for targeting peroxisomal ascorbate peroxidase (pAPX) to glyoxysomes, and define the topographic orientation of the protein within the membrane. This information is particularly relevant for oilseed glyoxysomes, to understand how the membrane is protected from abundant reactive oxygen species and to understand the pathway for regeneration of NAD+ required for conversion of storage oils into carbohydrates. The second aim is to test the hypothesis that a 73 kilodalton membrane-bound polypeptide, PMP73, is a novel "peroxin" which functions at the boundary membrane as a molecular chaperone in the uptake of proteins from the cytosol. The third aim is to try to discover additional plant PM Ps, using polyclonal rabbit antisera raised previously and already used successfully to discover PMP73 and pAPX. It is likely that, in addition to the overall goal of understanding peroxisomal biogenesis and function in developing oilseed seedlings, the results of these studies will provide more general insight into peroxisomal biogenesis that will be applicable to diverse species and tissues.

Title: "Arabidopsis 2010: Plant Peroxisomal Biogenesis - Sorting/Function of Membrane Proteins and Peroxins".

Peroxisomes are ubiquitous subcellular organelles that possess a diverse array of enzymes and other protein contents that vary with developmental stage, and in response to environmental cues. The overall goal of this project is to identify Arabidopsis genes coding for peroxisomal membrane proteins (PMPs) involved in the biogenesis and functioning of peroxisomes, and then determine the specific function of the gene products. "Peroxin" genes code for a set of proteins (peroxins) that participate specifically in peroxisomal biogenesis (organelle replication and differentiation). Most peroxins are PMPs. To date, 15 Arabidopsis orthologs of 23 eukaryotic peroxin genes have been identified, but a function has been determined for only 4 of these 15 orthologs. Public access to this information will be available at (http://lsweb.la.asu.edu/rtreleaseq). The function(s) of these PMPs will be determined experimentally through a multi-pronged approach, i.e., elucidation of their subcellular localization, intracellular sorting pathways, and molecular sorting signals in suspension cells from wild type and mutant plants, and developmentally through interactions of RNAs with increasingly available ESTs on microarrays. Also, phenotypic and functional complementation results obtained with available T-DNA knockouts will be examined. Experimental results will be presented by students and postdoctoral persons attending local and national meetings, and will be published in refereed journal articles. The research is expected to elucidate the function of at least 10 genes related to the biogenesis and functioning of peroxisomes in Arabidopsis; this is consistent with the Objectives of the 2010 Project. Since peroxisomal mutations are lethal in humans, and peroxisomes are essential for seedling establishment and photo-autotrophic growth of oilseed and other crop plants, the knowledge obtained for functions of Arabidopsis peroxisomal genes can and very likely will be applied to biotechnological improvements of agriculturally-important crop plants, and to possibly speed therapeutic resolution of peroxisomal biogenetic diseases in human infants.