Role of Tim50 in the Transfer of Precursor Proteins from the Outer to the Inner Membrane of Mitochondria

Abstract

Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50 depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23 complexes requires the concerted action of Tim50 with Tim23.

Direct Interaction of Mitochondrial Targeting Presequences with Purified Components of the TIM23 Protein Complex

“…In the IMS, the precursor protein binds to Tim50 and Tim23, each with a low affinity. This step is followed by the formation of a ternary complex of Tim50-Tim23-precursor that was demonstrated to be essential for maintaining the interaction of precursor proteins with these components in vivo (7). At this stage, opening of the channel exposes the imported presequence to the pulling effect of the membrane potential.…”

“…These proteins form a channel that allows proteins to cross, or integrate into the inner mitochondrial membrane. An additional subunit, Tim50, serves as a receptor in the mitochondrial intermembrane space (IMS) and has a role in maintaining the permeability barrier of the channel (7)(8)(9)(10)(11). Together, these three proteins constitute the cis side of the inner membrane channel.…”

“…Tim50 could be cross-linked to various preproteins transiting the import channel, and therefore the function of a receptor was attributed to this subunit (7, 9 -11). Another component on the cis side, the soluble domain of Tim23, was suggested to receive precursor proteins from Tim50 and to transfer them further into the channel (7,(22)(23)(24). On the trans side of the membrane, Tim44 could be cross-linked to precursor proteins (25)(26)(27)(28)(29).…”

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

“…We thus analysed mutant mitochondria, which are impaired in subunits of the TIM23 complex [29][30][31][32]. Conditional mutants of the essential subunits Tim23, Tim50 and Tim17 were considerably impaired in the import of Om45 (Fig 3B and C).…”

Mitochondrial protein homeostasis

“…2). The first subunit of the TIM23 complex encountered by an incoming precursor protein on the trans-side of the TOM complex is Tim50 (26)(27)(28)(29). Tim50 passes the presequence to the channel forming unit of TIM23 formed by the Tim23 and Tim17 proteins.…”