INTERACTION OF MITOCHONDRIAL TARGETING SIGNALS WITH ACIDIC RECEPTOR DOMAINS ALONG THE PROTEIN IMPORT PATHWAY - EVIDENCE FOR THE ACID CHAIN HYPOTHESIS

Mitochondrial precursor proteins with basic targeting signals may be t ransported across the outer membrane by sequential binding to acidic r eceptor sites of increasing affinity. To test this 'acid chain' hypoth esis, we assayed the interaction of mitochondrial precursors with thre e acidic receptor domains: the cytosolic domain of Tom20 and the inter membrane space domain of Tom22 and Tim23, The apparent affinity and sa lt resistance of precursor binding increased in the order Tom20 < Tom2 2 (internal) < Tim23. Precursor binding to the three acidic receptor d omains and to the pure cytosolic domain of Tom70 was inhibited by exce ss targeting peptide, but not by an equally basic control peptide, In this membrane-free and defined system, a precursor pre-bound to the To m70 or Tom20 domain was transferred efficiently to the Tim23 domain. T ransfer was stimulated by the internal Tom22 domain and was much less efficient in the reverse direction. Precursors destined for the outer membrane bound only to Tom20, but not to the internal Tom22 or the Tim 23 domain, and a precursor destined for the inner membrane bound only to the Tom20 and the internal Tom22 domain, but not to the Tim23 domai n. These results suggest that specific and sequential binding of a tar geting signal to strategically situated acidic receptors delivers a pr ecursor across the outer membrane and contributes to intramitochondria l sorting of imported proteins.