Degree Type

Dissertation

Date of Award

2008

Degree Name

Doctor of Philosophy

Department

Genetics, Development and Cell Biology

First Advisor

Martin H. Spalding

Abstract

A Chlamydomonas gene CAH1 is responsible for the expression of the secreted protein periplasmic Carbonic Anhydrase 1 (CAH1), which undergoes unique post-translational processing. The pre-protein that has endomembrane targeting sequence and one large and one small subunit separated by a small 35 amino acid stretch called as spacer, undergoes proteolytic processing that cleaves off the targeting sequence and the spacer, giving rise to a mature, glycosylated CAH1 protein. This hetero-tetramer consists of two large and two small subunits attached to each other by disulphide bridges.

This research work involves the analysis of the intrinsic factors of the pre-protein that are essential for the proteolytic cleavage of the spacer from the pre-protein. Various potential target sites for the proteases, in and around the spacer region for the proteolytic cleavage were investigated by generating transgenic Arabidopsis plants expressing mutated forms of CAH1 gene. Failure to locate any amino acid sequence/s inside or around the spacer region that controls proteolysis suggests that the proteolysis of the pre-protein is largely independent of the sequence of amino acid residues separating large and small subunits which was supported by successful demonstration of proteolysis of the constructs with the spacer with reverse order of amino acid residues and the spacer with radically different amino acid sequence, in transgenic Arabidopsis plants. We predict possible involvement of more than one protease that act on more than one target sequence in and/or around the spacer region. Analysis of the constructs with progressively reducing number of amino acid residues between the disulfide bond forming cystine residues of large and small subunit suggests that the proteolytic cleavage may be dependent more on the number and the composition of the amino acids between the two cystine residues that form a disulfide bridge between large and small subunits, than the actual sequence. Most interestingly the successful proteolysis of the CAH1 construct lacking previously demonstrated spacer region questions the very requirement of the spacer for the proteolysis of the pre-protein. Even though more research is required to understand this processing better, the post-translational processing undergone by CAH1 may utilize a mechanism that is conserved across the diverse kingdoms making it an interesting and important process to study.

Copyright Owner

Parijat S. Juvale

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

81 pages

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