Functional analysis of the membrane bound aminopeptidase, APM1
Abstract
The purpose of the present study was to investigate the role of the M1 metallopeptidase APM1 in plant development. APM1 has been shown to be strongly expressed in the shoot apex, young leaves, and root vascular tissue of Arabidopsis and is essential for normal embryonic, vegetative, and reproductive development. Analyses of the apm1 mutants revealed that there is a collapsed quiescent center, decreased differentiation of the root vascular tissue, and alteration in cell division and radial patterning in apm1 roots. Inducible expression studies demonstrate that APM1 is required at two stages for root and shoot development. Like other members of a subclass of M1 peptidases, APM1 has a catalytic domain an interaction domain, both of which contribute to its function. Treatment of Arabidopsis seedlings with ezetimibe, which targets M1 protein/protein interactions, mimics a subset of apm1 phenotypes distinct from those resulting from treatment with the M1/APM1 catalytic inhibitor PAQ-22. Site-directed mutagenesis showed that the catalytic domain is necessary for APM1 function. The protein interactions that confer activity appear to be dependent on the C-terminal region of the APM1, as transformation with an open reading frame in which the last 159 aa residues were deleted did not restore wild type growth to apm1-1. Further, multiple amino acid interactions appear to be required for APM1 function. Internal deletion of a portion of the C-terminus partially inhibited APM1 function. Transformation of apm1-2, which contains a functional enzymatic domain but lacks the last 211 aa residues of the protein, catalytically dead APM1 restores wild type growth. These results suggest that both the catalytic and interaction domains are necessary for APM1 function throughout development, but that protein function does not require the domains to be physically linked. APM1 functions more in a PSA/APN-like manner rather than in an IRAP-like manner and that the membrane localization of APM1 is important to its activity, without which, the dimer may not form thereby preventing APM1 from being targeted to its appropriate destination.
Degree
Ph.D.
Advisors
Murphy, Purdue University.
Subject Area
Plant biology
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