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| Author(s): |
Phellippe Arthur Santos Marbach
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Piracicaba. |
| Institution: | Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC) |
| Defense date: | 2004-04-30 |
| Advisor: | Márcio de Castro Silva Filho |
| Abstract | |
Metalloproteases AAA are ubiquitous in Bacteria and Eukarya domains, however they are not found in Archaeas. These enzymes are integral proteins involved in essential cellular processes such as oxidative phosphorylation, photosynthesis and organellar biogenesis. The Escherichia coli metalloprotease AAA named FtsH, is the most studied member of this protein. Analysis of the distribution of the AAA metalloprotease encoded genes in bacterial genomes revealed that the majority of the bacterial genomes contains only one copy of the metalloprotease gene. The existence of more than one FtsH isoform per genome seems to be limited to some bacterial groups. Phylogenetic analysis of AAA metalloproteases showed the existence of three distinct isoforms, resulting from old duplication events. These isoforms show high similarity at the primary and secondary structural level. Phylogenetic analysis of eukaryotic AAA metalloproteases showed that they can be clustered into five clades. Members of the same clade were predicted to have the same subcellular localization, plastid or mitochondria. The plastidial clades were named FtsHp1, FtsHp2 e FtsHp3, and the mitochondrial clades FtsHm1 e FtsHm2. The distribution of metalloproteases AAA in the domains of life as well as their predicted subcellular localization, suggested an endosymbiotic origin of the eukaryotic members. In fact, the phylogenetic, analysis including both bacterial and eukaryotic AAA metaloproteases, showed an ancient relationship among cyanobacterial e plastidial-derived AAA metaloproteases. The same observation was shown for the FtsHm1 metaloproteases clade and the ancestor of actual proteobacterias. Surprisingly, this phylogenetic analysis sugest tat the FtsHm2 clade ancestor was originated from a horizontal gene transfer event between the primitive eukaryotic cell and a species from CFB bacterial division. The subcellular localization of sugarcane FtsHp1, FtsHp2 e FtsHm1 were studied to test the hypothesis that the phylogenetic profile of the plant metaloproteases AAA can be used to predict their subcellular localization. Gene fusion involving the targeting sequence of these proteins and CFP (Green Fluorescent Protein) were expressed in onion epidermal cells and the subcellular localization of GFP was monitored with a fluorescent microscope. The GFP fused to the FtsHp1 e FtsHp2 targeting sequences was targeted to plastids while the GFP fused to the FtsH-m1 targeting sequence of was exclusively targeted to mitochondria. These results confirm the hypothesis that the subcellular localization of the plant AAA metaloproteases can be predicted based on their phylogenetic profile. (AU) | |