The phosphate carrier from yeast mitochondria. Dimerization is a prerequisite for function.
Wild type phosphate carrier (PIC) from Saccharomyces cerevisiae and recombinant PIC proteins with different C-terminal extensions were expressed in Escherichia coli as inclusion bodies. From these, PIC was isolated with the detergent sodium lauroyl sarcosinate in a form, partially monomeric and unfolded. This PIC associates to stable dimers after exchanging the detergent to the polyoxyethylene detergent C12E8 and dialysis. Combining two differently tagged monomers of PIC and following this with affinity chromatography yields defined homo- and heterodimeric forms of PIC, which are all fully active after reconstitution. As a member of the mitochondrial carrier family PIC is supposed to function as a homodimer. We investigated its dimeric nature in the functionally active state after reconstitution. When reconstituting PIC monomers a sigmoidal dependence of transport activity on the amount of inserted protein is observed, whereas insertion of PIC dimers leads to a linear dependence. Heterodimeric PIC constructs consisting of both an active and an inactivated subunit do not catalyze phosphate transport. In contrast, reconstitution of a mixture of active and inactive monomeric subunits led to partially active carrier. These experiments prove (i) that PIC does not function in monomeric form, (ii) that PIC dimers are stable both in the solubilized state and after membrane insertion, and (iii) that transport catalyzed by PIC dimers involves functional cross-talk between the two monomers.
Schroers
A
A
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Burkovski
A
A
Wohlrab
H
H
Krämer
R
R
D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry*; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000502:physiology; D008928:Mitochondria / Q000502:physiology*; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000502:physiology*
D002352:Carrier Proteins; D003902:Detergents; D005656:Fungal Proteins; D008081:Liposomes; D008565:Membrane Proteins; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D010743:Phospholipids; D011994:Recombinant Proteins; D005033:Ethylmaleimide