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(7) FUNCTIONAL MODIFICATIONS OF THE PROTEIN TRANSLOCATION CHANNEL INDUCED BY GENETIC ALTERATIONS OF THE TIM COMPLEX M.L. Campo1, S. Martinez-Caballero1,
A. Gázquez1 and K.W. Kinnally2
Water-filled channels are central to the process of translocating proteins since they provide aqueous pathways through the hydrophobic environment of membranes. The TOM and TIM complexes translocate precursors across the mitochondrial outer and inner membranes, respectively. Using electrophysiological techniques we have identified the channel that purportedly functions in the TIM complex. In an attempt to identify if some of the known components of this complex might function as the actual pore, we are undertaking several structure-function studies. Inner membranes purified from mitochondria of different mutants of Sacharomyces cerevisiae were reconstituted into proteoliposomes and the electrophysiological properties of the channels were studied by patch-clamping. Our recent results indicate reduced expression of Tim17p modifies the channel behavior. The mean open time is significantly longer that of the control and the voltage dependence is disrupted. While signal sequence peptides typically induce a rapid flickering of the wild type channel, these peptides cause an immediate 500 pS closure of the channel from the Tim17p depletion mutant. Furthermore, this singular peptide sensitivity differs from that obtained by depletion of Tim23p, a receptor of TIM complex. These findings suggest that Tim17p may be responsible for the gating properties of the translocation channel of the mitochondrial inner membrane. This work was supported by DGICYT grant PB98-0988 and Junta de Extremadura & Fondo Social Europeo grant IPR99 C003 to MLM, SMC and AG, and NSF grants MCB-9513439, MCB-9816950 and MCB-0096206 to KWK.
For further information contact...Carmen Mannella: carmen@wadsworth.org |
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