A novel growth-related AAA ATPase from plants

Carrie Allison Hicks, Purdue University

Abstract

The process of cell enlargement whereby cells enlarge following cytokinesis has been little studied. The purpose of this thesis was to develop a cell-free assay which would facilitate the study of this phenomenon and to identify involved genes and gene products. Using plants, a novel cell-free vesicle enlargement assay was developed which mimics auxin growth factor-induced cell enlargement Findings show that vesicle and cell enlargement are the result of an active and energy-requiring process. Isolated inside-out but not right side-out soybean plasma membrane vesicles nearly double in volume in an ATP-dependent manner when incubated together with active auxins. The kinetics of auxin-induced enlargement of vesicles approximates the kinetics of auxin-stimulated cell enlargement in vivo. The ATP + 2,4-D-dependent vesicle enlargement has a pH optimum of 8.5, nucleotide specificity for ATP, does not occur in the presence of ATP-γ-S, has a Km of 50μM, suggesting that enlargement is an ATPase-driven process. Typical ATPase inhibitors of the vacuolar-(KNO3), Na2+-(ouabain), K+-(oligomycin), and the H+-ATPases (vanadate), do not inhibit ATP + 2,4-D-induced vesicle enlargement. N-ethylmaleimide and COCl2 do inhibit vesicle enlargement and are known to inhibit AAA-ATPase, ATPases Associated with a variety of cellular Activites. The ATP-dependent component of auxin-induced vesicle enlargement was identified as an AAA-ATPase. Two anti-AAA-ATPase polyclonal peptide antibodies that inhibit cell-free vesicle enlargement were used to screen a soybean cDNA expression library. A 2680 bp gene was cloned which encodes 807 amino acids corresponding to 87 kD. The protein contains two copies of a highly conserved 200 amino acid ATP-binding module which is a defining characteristic of the AAA-ATPases. The 87 kD soybean AAA-ATPase protein was isolated and demonstrated to be associated with the plasma membrane. It has ATPase activity but is not auxin-responsive. The findings are interpreted to suggest that plant cell enlargement results from an active ATP-driven displacement of cell surface membrane constituents rather than a passive turgor-driven yielding of auxin-loosened cell walls. The essential ATPase has been identified as a novel plasma membrane associated AAA-ATPase that most likely functions in concert with other membrane components involved in auxin-induced cell enlargement.

Degree

Ph.D.

Advisors

Morre, Purdue University.

Subject Area

Botany|Cellular biology|Molecular biology|Biochemistry

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