Protein Import by the Mitochondrial Presequence Translocase in the Absence of a Membrane Potential

• Published in: Journal of molecular biology Vol. 428; no. 6; pp. 1041 - 1052
• Main Authors: Turakhiya, Uma, von der Malsburg, Karina, Gold, Vicki A.M., Guiard, Bernard, Chacinska, Agnieszka, van der Laan, Martin, Ieva, Raffaele
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 27.03.2016
• Subjects: ATP synthase; DNA Mutational Analysis; membrane potential; Membrane Potentials; Membrane Transport Proteins - metabolism; Mitochondria - metabolism; Mitochondria - physiology; mitochondrial biogenesis; Mitochondrial Proton-Translocating ATPases - genetics; Mitochondrial Proton-Translocating ATPases - metabolism; protein import; Protein Transport; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; TIM23 complex; Su e; DHFR; CCCP; ATP synthase; membrane potential; protein import; mitochondrial biogenesis; Su g; TIM23 complex
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2016.01.020

The highly organized mitochondrial inner membrane harbors enzymes that produce the bulk of cellular ATP via oxidative phosphorylation. The majority of inner membrane protein precursors are synthesized in the cytosol. Precursors with a cleavable presequence are imported by the presequence translocase (TIM23 complex), while other precursors containing internal targeting signals are imported by the carrier translocase (TIM22 complex). Both TIM23 and TIM22 are activated by the transmembrane electrochemical potential. Many small inner membrane proteins, however, do not resemble canonical TIM23 or TIM22 substrates and their mechanism of import is unknown. We report that subunit e of the F1Fo-ATP synthase, a small single-spanning inner membrane protein that is critical for inner membrane organization, is imported by TIM23 in a process that does not require activation by the membrane potential. Absence of positively charged residues at the matrix-facing amino-terminus of subunit e facilitates membrane potential-independent import. Instead, engineered positive charges establish a dependence of the import reaction on the electrochemical potential. Our results have two major implications. First, they reveal an unprecedented pathway of protein import into the mitochondrial inner membrane, which is mediated by TIM23. Second, they directly demonstrate the role of the membrane potential in driving the electrophoretic transport of positively charged protein segments across the inner membrane. [Display omitted] •A novel pathway of import for small proteins of the inner mitochondrial membrane has been discovered.•This pathway is mediated by the TIM23 import machinery and it does not require translocase activation by ∆ψ in isolated mitochondria.•Lack of positive charges in the matrix domain of subunit e facilitates precursor import in the absence of ∆ψ.