Isolation and structural characterization of a subset of yeast (Saccharomyces cerevisiae) peroxisomal proteins
Nandi, Munmun S
MetadataShow full item record
Peroxisomes are virtually found in all eukaryotic cells, but unlike mitochondria and chloroplasts, they do not contain DNA or a protein secretory apparatus. Therefore, all of their proteins must be imported by a process called peroxisomal biogenesis. This requires a group of protein factors referred to as peroxins which are encoded by the pex genes. Currently, there are approximately thirty-two known peroxisomal proteins. Among all the peroxisomal proteins, two enzymes namely GPD1, LYS1 and a peroxin, PEX7 were selected for the research. GPD1 is a NAD+ -dependent glycerol 3-phosphate dehydrogenase1 that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate which is crucial for growth under osmotic stress. Its purification was achieved using ion exchange chromatography and the pure protein was crystallized for structure determination. Diffraction data sets were obtained to a resolution of 2.2 Å which were used to solve the C-terminal portion of the structure. Unfortunately, the N-terminal portion remained disordered. LYS1 is the terminal enzyme of α-aminoadipate pathway and catalyzes the reversible NAD-dependent oxidative cleavage of saccharopine to yield L-lysine and α-ketoglutarate. The purification of LYS1 was carried out using affinity chromatography. Another protein, PEX7 is responsible for peroxisome biogenesis by importing newly synthesized proteins bearing PTS2 (peroxisome targeting signal sequence2) into peroxisomes. PEX7 presented the greatest challenge among the three proteins at both the expression stage and the purification stage. Its soluble fraction was purified using ion exchange and affinity chromatographies, although the final yield was too low for crystallization trials. A much large proportion of the protein was found in the insoluble cell debris and attempts were made to purify this fraction after denaturation. An alternative, protocol involving the formation of a GPD1-PEX7 complex proved to be effective route to co-purification of the two proteins and crystallization trials are proceeding. Having known the structures of peroxisomal proteins, it would be helpful for studying the development and maintenance of the organelle related to its metabolic diseases in the eukaryotic cells.