Substitutional mutations in the uncoupling protein-specific sequences of mitochondrial uncoupling protein UCP1 lead to the reduction of fatty acid-induced H+ uniport.
Mutants were constructed for mitochondrial uncoupling protein UCP1, with single or multiple substitutions within or nearby the UCP-signatures located in the first alpha-helix and second matrix-segment, using the QuickChange site directed mutagenesis protocol (Stratagene), and were assayed fluorometrically for kinetics of fatty acid (FA)-induced H+ uniport and for Cl- uniport. Their ability to bind 3H-GTP was also evaluated. The wild type UCP1 was associated with the FA-induced H+ uniport proportional to the added protein with a Km for lauric acid of 43 micro M and Vmax of 18 micro molmin(-1)(mg protein)(-1). Neutralization of Arg152 (in the second matrix-segment UCP-signature) led to approximately 50% reduction of FA affinity (reciprocal Km) and of Vmax for FA-induced H+ uniport. Halved FA affinity and 70% reduction of Vmax was found for the double His substitution outside the signature (H145L and H147L mutant). Neutralization of Asp27 in the first alpha-helix UCP-signature (D27V mutant) resulted in 75% reduction of FA affinity and approximately 50% reduction of Vmax, whereas the triple C24A and D27V and T30A mutant was fully non-functional (Vmax reduced by 90%). Interestingly, the T30A mutant exhibited only the approximately 50% reduced FA affinity but not Vmax. Cl- uniport and 3H-GTP binding were preserved in all studied mutants. We conclude that amino acid residues of the first alpha-helix UCP signature may be required to hold the intact UCP1 transport conformation. This could be valid also for the positive charge of Arg152 (second matrix-segment UCP signature), which may alternatively mediate FA interaction with the native protein.
Urbánková
Eva
E
Department of Membrane Transport Biophysics, No. 375, Institute of Physiology, Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic.
Hanák
Petr
P
Skobisová
Eva
E
Růzicka
Michal
M
Jezek
Petr
P
D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism* / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D007473:Ion Channels; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D007850:Lauric Acids / Q000494:pharmacology; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D016297:Mutagenesis, Site-Directed; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D002712:Chlorides; D005227:Fatty Acids; D007473:Ion Channels; D007850:Lauric Acids; D008081:Liposomes; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; C000604373:Ucp1 protein, rat; D000071256:Uncoupling Protein 1; C030358:lauric acid; D006859:Hydrogen; D006160:Guanosine Triphosphate