Mitochondrial decoupling protein from plants, PUMP: Calorimetric studies and functionality of Arabidopsis thaliana PUMP expressed as E. coli

Author(s)

Paula Bresciani Martins de Andrade

• Document type: Doctoral Thesis
• Press: São Paulo. , gráficos, ilustrações, tabelas.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2002-09-04

Examining board members

Hernan Chaimovich Guralnik; Roger Frigerio Castilho; Rui Curi; Luis Eduardo Soares Netto; Shirley Schreier; Bayardo Baptista Torres

• Advisor: Hernan Chaimovich Guralnik
• Field of knowledge: Biological Sciences - Biochemistry
• Indexed in: Banco de Dados Bibliográficos da USP-DEDALUS
• Location: Universidade de São Paulo. Biblioteca do Conjunto das Químicas; CQ T/581.19245; A553p

Abstract

In 1995, a plant mitochondrial uncoupling protein, PUMP, was first described. PUMP, like the known uncoupling protein from brown adipose tissue, UCP1, increases the inner mitochondrial membrane permeability to H+. H+ transport is dependent on the presence of free fatty acids, FFA, and it is inhibited by purine nucleotides, PN. PUMP expression and activity are stimulated by cold exposure, which may vary during fruit ripening. By expressing a full length cDNA encoding the Arabidopsis UCP in E. coli, the recombinant AtPUMP was obtained. In this study, AtPUMP was incorporated in proteoliposomes and the functionality of the protein was demonstrated. The incorporation of uncoupling proteins in proteoliposomes constitutes a model that allows the functional and the mechanistic analysis of these proteins. The H+ conductance mediated by reconstituted potato PUMP was, undoubtedly, activated by FFA. However, the inhibition by PN was not reproducible. Reconstituted AtPUMP was activated by FFA and the apparent Km\'s were determined: 42 µM (linoleic acid, LA), 55 µM (lauric acid) and 70 µM (palmitic acid). Reconstituted AtPUMP was inhibited by PN, and the apparent Kis were determined: 0.8 mM (GDP), 0.85 mM (ATP), 0.98 mM (GTP) and 1.4 mM (ADP). AtPUMP mediated H+ efflux rate, activated by LA, was exponentially dependent on membrane potential (Δψ). The partition coefficient (KP) between the aqueous phase and the membrane phase was determined for LA. The KP was 1.6 times higher for AtPUMP proteoliposomes than for liposomes. Using Isothermal Titration Microcalorimetry (ITC), we verified that there is a linear correlation between the heat produced and oxygen depletion (65,2 kcal/mol O2) in a suspension of potato mitochondria, when PUMP was activated by LA. Using ITC we also determined that LA might bind to two different sites in AtPUMP. Based on the results obtained, we concluded that PUMP (or AtPUMP) is a plant mitochondrial uncoupler. The reconstitution assays permitted the study of FFA and PN regulation. In addition, our results represent the first direct confirmation that fatty acids regulate heat release in plant mitochondria, a process that may play a role in cold adaptation, fruit ripening and flower blossoming. (AU)