The sorting signal of cytochrome b2 promotes early divergence from the general mitochondrial import pathway and restricts the unfoldase activity of matrix Hsp70.
Cytochrome b2 is imported into mitochondria and sorted to the intermembrane space by a bipartite N-terminal presequence, which is a matrix targeting sequenced followed by an intermembrane space sorting signal. The N-terminus of the mature protein forms a folded heme binding domain that depends on the unfoldase function of matrix (mt) Hsp70 for import. We report that the distance between the presequence and the heme binding domain is critical for the ability of mt-Hsp70 to promote import of the domain. Hybrid proteins with 40 or more amino acids between the presequence and the heme binding domain are arrested in the import machinery. The translocation arrest can be overcome by unfolding of the preprotein or by inactivation of the intermembrane space sorting signal. Moreover, the sorting signal prevents backsliding of the precursor polypeptide in the import site in the initial import step, when the signal has not made contact with the matrix. The results indicate that the sorting signal interacts with component(s) of the inner membrane/intermembrane space during the initial import step and promotes an early divergence of b2 preproteins from the general matrix import pathway, precluding an unfolding role for mt-Hsp70 in the translocation of most of the mature portions of a preprotein. We propose a sorting model of cytochrome b2 which explains the apparently divergent previous results by a unifying hypothesis.
Gärtner
F
F
Biochemisches Institut, Universität Freiburg, Germany.
Bömer
U
U
Guiard
B
B
Pfanner
N
N
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D021382:Protein Sorting Signals / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D006418:Heme; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D013762:Tetrahydrofolate Dehydrogenase