THE INTERMEMBRANE SPACE DOMAIN OF MITOCHONDRIAL TOM22 FUNCTIONS AS A TRANS BINDING-SITE FOR PROPERTIES WITH N-TERMINAL TARGETING SEQUENCES

Mitochondrial protein impart is thought to involve the sequential inte raction of preproteins with binding sites on cia and trans sides of th e membranes, For translocation across the outer membrane, preproteins first interact with the cytosolic domains of import receptors (cis) an d then are translocated through a general import pore, in a process pr oposed to involve binding to a trans site on the intermembrane space ( IMS) side. Controversial results have been reported for the role of th e IMS domain of the essential outer membrane protein Tom22 in formatio n of the trans site, We show with different mutant mitochondria that a lark of the IMS domain only moderately reduces the direct import of p reproteins with N-terminal targeting sequences. The dependence of impo rt on the IMS domain of Tom22 is significantly enhanced by removing th e cytosolic domains of import receptors ok by performing import in two steps, i.e., accumulation of a preprotein at the outer membrane in th e absence of a membrane potential (Delta psi) and subsequent import af ter reestablishment of a Delta psi. After the removal of cytosolic rec eptor domains, two-step import of a cleat-able preprotein strictly req uires the IMS domain. In contrast, preproteins with internal targeting information do not depend on the IMS domain of Tom22, We conclude tha t the negatively charged IMS domain of Tom22 functions as a traits bin ding site for preproteins with N-terminal targeting sequences, in agre ement with the acid chain hypothesis of mitochondrial protein import.