The channel-forming Sym1 protein is transported by the TIM23 complex in a presequence-independent manner

Abstract

The majority of multispanning inner mitochondrial membrane proteins utilize internal targeting signals, which direct them to the carrier translocase (TIM22 complex), for their import. MPV17 and its Saccharomyces cerevisiae orthologue Sym1 are multispanning inner membrane proteins of unknown function with an amino-terminal presequence that suggests they may be targeted to the mitochondria. Mutations affecting MPV17 are associated with mitochondrial DNA depletion syndrome (MDDS). Reconstitution of purified Sym1 into planar lipid bilayers and electrophysiological measurements have demonstrated that Sym1 forms a membrane pore. To address the biogenesis of Sym1, which oligomerizes in the inner mitochondrial membrane, we studied its import and assembly pathway. Sym1 forms a transport intermediate at the translocase of the outer membrane (TOM) complex. Surprisingly, Sym1 was not transported into mitochondria by an amino-terminal signal, and in contrast to what has been observed in carrier proteins, Sym1 transport and assembly into the inner membrane were independent of small translocase of mitochondrial inner membrane (TIM) and TIM22 complexes. Instead, Sym1 required the presequence of translocase for its biogenesis. Our analyses have revealed a novel transport mechanism for a polytopic membrane protein in which internal signals direct the precursor into the inner membrane via the TIM23 complex, indicating a presequence-independent function of this translocase.

Fig 1

Isolation of Sym1 from mitochondrial membranes. (A) ClustalW2 sequence alignment of Sym1 homologues in fungi and mammals. Black boxes indicate 100% similarity, and gray boxes indicate 75% similarity. (B) Schematic representation of the ZZ tag fused to Sym1 (ZZ, two repeats of the Z domain of protein A; TEV, cleavage site of the tobacco etch virus protease; His₁₀, decahistidine tag). The epitope recognized by the Sym1 antibody is indicated (αSym1). IMS, intermembrane space; MIM, mitochondrial inner membrane. (C) Isolated mitochondria from the wild type (WT) and the SYM1^ZZ strain were subjected to SDS-PAGE and Western blotting using antibodies against Sym1 and PAP (HRP–anti-HRP). (D) Mitochondria were left untreated or subjected to swelling in hypo-osmotic buffer, treated with the indicated amounts of protease, and subjected to SDS-PAGE and Western blotting. Sym1^ZZ was detected using PAP. Prot. K, proteinase K. (E) Flow diagram of the isolation strategy for Sym1^ZZ. (F) Total (5%), unbound (UB) (5%), and eluate (E) (E_Ni, 5%; E_IgG, 100%) fractions of Ni-NTA affinity and IgG affinity chromatography were separated by SDS-PAGE and subjected to Western blot analyses using the Sym1 antibody. (G) Coomassie brilliant blue-stained SDS-PAGE gel of the IgG chromatography eluate.

Fig 2

Sym1 forms a channel after reconstitution into planar lipid bilayers. (A) Sym1-containing proteoliposomes were fused with lipid bilayers, and single-channel activity was recorded in 250 mM KCl; (B) conductance state histograms deduced from gating events at positive and negative membrane potentials; (C) current-voltage relationship under asymmetrical buffer conditions (U_rev, reversal potential); (D) voltage-dependent open probability (Popen); (E) current-voltage relationship under symmetrical buffer conditions in the presence or absence of a specific antibody against Sym1; (F) conductance states in the presence or absence of an anti-Sym1 antibody.

Fig 3

Sym1 is directed to mitochondria by internal targeting signals. (A) Schematic representation of the transmembrane (TM) domains of Sym1 and inner membrane channels of similar topology. (B) Radiolabeled MPV17 and SURF1 were imported into mitochondria isolated from HEK293T cells. In vitro import was performed for the indicated times in the presence or absence of a membrane potential (Δψ). Reisolated mitochondria were treated with proteinase K (Prot. K) where indicated and analyzed by SDS-PAGE and digital autoradiography. p, precursor; m, mature. (C) ³⁵S-labeled proteins were imported into isolated yeast mitochondria and analyzed by SDS-PAGE and digital autoradiography. (D) Constructs for analysis of targeting signals. FL, full-length protein; ΔN, N-terminal truncation; ΔC, C-terminal truncation; TM, transmembrane span. (E) In vitro import of truncation constructs into isolated yeast mitochondria as described for panel C. Indicated samples were sonicated in the presence or absence of proteinase K. Truncated Sym1s originating from a second start codon in SYM1 are indicated with asterisks. (F) Carbonate extraction of mitochondrial membranes at the indicated pH. After centrifugation, total fractions of samples (T), pellets (P), and supernatants (S) were analyzed by Western blotting.

Fig 4

Assembly of Sym1 into mature complexes. (A) Yeast mitochondria were solubilized in digitonin buffer, separated by BN-PAGE, and analyzed by Western blotting using the PAP antibody; (B) Sym1^ZZ isolated from mitochondria by IgG chromatography was analyzed by BN-PAGE and stained with Coomassie brilliant blue; (C) mitochondria were solubilized in digitonin-containing buffer, and complexes were separated by 1st-dimension BN-PAGE and 2nd-dimension SDS-PAGE and analyzed by Western blotting with the anti-Sym1 antibody; (D) mitochondria were solubilized in buffers containing digitonin (Dig.) or dodecyl maltoside (DDM), and the complexes were separated by 1st-dimension BN-PAGE and 2nd-dimension SDS gel and analyzed by Western blotting; (E) Sym1 import was monitored with BN-PAGE and subsequent digital autoradiography; (F) import and BN-PAGE analysis of truncation constructs described for Fig. 3D. Complexes were visualized by digital autoradiography.

Fig 5

TOM complex intermediates formed during Sym1 import. (A) Analysis of [³⁵S]Sym1 and [³⁵S]AAC import into mitochondria without proteinase K treatment; (B) comparison of short-time import of [³⁵S]Sym1 proteinase K-treated and nontreated samples; (C) mitochondria were solubilized after import of [³⁵S]Sym1 for 3 min and incubated with the indicated antibodies before separation by BN-PAGE and digital autoradiography; (D) radiolabeled Sym1 was imported into tom22^His mitochondria. After solubilization, Tom22^His-containing complexes were eliminated by incubation with Ni-NTA beads. Total (T) and unbound (U) fractions were separated by BN-PAGE and analyzed by digital autoradiography.

Fig 6

Sym1 is imported independent of Tim9/Tim10 and TIM22 complexes. Radiolabeled proteins were imported into mitochondria isolated from the WT and the tim10-2 (A), tim54-11 (B) and tim12-4 (C) mutant strains. In vitro import was performed for the indicated times in the presence or absence of a membrane potential (Δψ). Reisolated mitochondria were solubilized in digitonin and analyzed by BN-PAGE and digital autoradiography.

Fig 7

Sym1 is imported by the TIM23 translocase. (A and B) Import of radiolabeled proteins into isolated WT and mutant mitochondria (as indicated) and analysis by BN-PAGE and digital autoradiography. (C) [³⁵S]F1β (squares) and [³⁵S]Su9-DHFR (triangles) were imported into isolated mitochondria (Tim50^HA, filled symbols; Tim50^ΔPBD-HA, open symbols) and analyzed by SDS-PAGE followed by digital autoradiography (left) and quantification (right). (D) ³⁵S-labeled proteins were imported into WT and mutant mitochondria and analyzed as described for panels A and B. (E and F) Quantification of the Sym1 120-kDa and 220-kDa complexes shown in panels B and D, respectively, comparing import into wild-type (WT) (filled symbols) and mutant (open symbols) mitochondria.

Fig 8

Schematic presentation of import substrates using the TIM23 complex. PAM, presequence translocase-associated protein import motor; MPP, mitochondrial processing peptidase; IM, inner mitochondrial membrane; IMS, intermembrane space.