Date of Award

1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Randall C. Gayda

Abstract

Cell division protein, FtsA, is an inner membrane protein with a putative ATP binding motif. Although FtsA has been studied genetically, it has not been characterized biochemically. Immunoelectron microscopy with FtsA polyclonal antibodies permitted FtsA to be within the cell. Since native FtsA is present in low amounts, the cellular level of this protein was increased by induction of ftsA from an overexpression plasmid before processing the cells for immunogold labelling. The immunogold particles localized FtsA to the inner cytoplasmic membrane. Immunogold labelling of cells overproducing C-terminal deleted FtsA, displayed the gold particles throughout the cytoplasm. This suggested that the C-terminal domain of FtsA may be the membrane anchor. In Western blots of proteins from membrane and soluble fractions obtained from wild-type cells, FtsA polyclonal antibody cross-reacting bands were found in both fractions. FtsA solubilized from membranes using Sarkosyl bound $\lbrack \alpha$-$\rm\sp{32}P\rbrack ATP.$ Urea-solubilized FtsA did not bind ATP under similar conditions. Sarkosyl-solubilized FtsA irreversibly aggregated during column purification attempts, and hence, Sarkosyl was exchanged with octylglucoside. FtsA was purified successfully in the presence of octylglucoside. This FtsA did not bind ATP. To obtain FtsA in a soluble and functional conformation, FtsA was co-expressed with chaperones, GroES and GroEL. FtsA was purified from the supernatant by successive anion exchange and nucleotide affinity columns. The FtsA fraction from an Affigel Blue column demonstrated $\lbrack \alpha$-$\rm\sp{32}P\rbrack ATP$ binding activity. But on further purification, only a dimer species of the purified FtsA bound $\lbrack \alpha$-$\rm\sp{32}P\rbrack ATP.$ This ATP binding was intrinsic to FtsA, and was abolished by EDTA, unlabeled ATP, heat or FtsA antibodies. The dimer form was barely detectable on SDS-PAGE where the monomeric FtsA band was distinctly seen. However, immunoblotting with anti-FtsA verified the band in the dimer region as FtsA. Western blot screening of the proteins from cell lysates of various eubacteria and unicellular eukaryotes for the presence of FtsA homologs revealed that all contained a major immunoreactive protein band. No FtsA band was found in cell wall-less Mycoplasma, consistent with FtsA being required for peptidoglycan biosynthesis.

ISBN

9780591905380

Pages

133

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