Import of cytochrome b₂ to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP

Abstract

Cytochrome b2 is synthesized as a precursor in the cytoplasm and imported to the intermembrane space of yeast mitochondria. We show here that the precursor contains a tightly folded heme-binding domain and that translocation of this domain across the outer membrane requires ATP. Surprisingly, it is ATP in the mitochondrial matrix rather than external ATP that drives import of the heme-binding domain. When the folded structure of the heme-binding domain is disrupted by mutation or by urea denaturation, import and correct processing take place in ATP-depleted mitochondria. These results indicate that (1) cytochrome b2 reaches the intermembrane space without completely crossing the inner membrane, and (2) some precursors fold outside the mitochondria but remain translocation-competent, and import of these precursors in vitro does not require ATP-dependent cytosolic chaperone proteins.

Two distinct mechanisms drive protein translocation across the mitochondrial outer membrane in the late step of the cytochrome b 2 import pathway

“…Residues 1-80 and residues 81-180 of the cytochrome b 2 part correspond to the presequence and the HBD, respectively. It is known that the efficient processing of the cytochrome b 2 presequence by Imp1p requires the presence of a correctly folded HBD that is stabilized by the bound heme group (11,12). We introduced mutations in the HBD to destabilize its folded conformation (Fig.…”

“…In both models, the translocation of the first part of the presequence across the inner membrane is coupled with the translocation of the HBD across the outer membrane and is driven by ⌬⌿ and ATP-dependent mHsp70 (11,13,14). However, once the tightly folded HBD has passed the outer membrane, the subsequent translocation of the rest of the polypeptide chain across the outer membrane becomes independent of ⌬⌿ and mHsp70 (11,13,14). The outer membrane lacks a transmembrane potential, and no ATP-dependent chaperone has been found in the IMS.…”

“…The first part of the presequence directs the protein to mitochondria and is proteolytically cleaved off by the mitochondrial processing peptidase in the matrix, generating an intermediate-size form, and the second part of the presequence mediates the transfer of the protein to the IMS and is removed by the inner-membrane protease I (Imp1p) on the IMS side of the inner membrane to yield a mature-size form. The mature part of cytochrome b 2 contains a heme-binding domain (HBD) in the N-terminal region (residues 81-180), which can fold independently of the rest of the molecule (11,12). The import pathway for the cytochrome b 2 precursor to the IMS has been a matter of debate for many years (4)(5)(6).…”

N-terminal Hydrophobic Sorting Signals of Preproteins Confer Mitochondrial hsp70 Independence for Import into Mitochondria

“…These data suggest that the cytochrome b 2 -derived preproteins follow the same sorting route irrespective of whether mt-hsp70 is functional or not. Only in the case of those cytochrome b 2 -derived proteins bearing an intact folded heme domain is their import dependent on mt-hsp70 intervention (28,36,37). We propose that the sorting signal enters the matrix, where it is specifically recognized and secured by a putative sorting signal recognition protein.…”

A model system for mitochondrial biogenesis reveals evolutionary rewiring of protein import and membrane assembly pathways

“…2A details the pathway for Cyb2 import via the stop-transfer sorting pathway as it occurs in S. cerevisiae. The import of ScCyb2 depends on active unfolding of the heme-binding domain by the action of matrixlocated Hsp70 (26)(27)(28)(29)(30). All the necessary components for the stop-transfer pathway are present in C. albicans (Table S1), but, as compared with S. cerevisiae, import of ScCyb2 is highly inefficient in mitochondria from C. albicans, with a greatly diminished processing to produce the intermediate ("iCyb2") and mature ("mCyb2") forms (Fig.…”