Detailed analysis of the human mitochondrial contact site complex indicate a hierarchy of subunits

Abstract

Mitochondrial inner membrane folds into cristae, which significantly increase its surface and are important for mitochondrial function. The stability of cristae depends on the mitochondrial contact site (MICOS) complex. In human mitochondria, the inner membrane MICOS complex interacts with the outer membrane sorting and assembly machinery (SAM) complex, to form the mitochondrial intermembrane space bridging complex (MIB). We have created knockdown cell lines of most of the MICOS and MIB components and have used them to study the importance of the individual subunits for the cristae formation and complex stability. We show that the most important subunits of the MIB complex in human mitochondria are Mic60/Mitofilin, Mic19/CHCHD3 and an outer membrane component Sam50. We provide additional proof that ApoO indeed is a subunit of the MICOS and MIB complexes and propose the name Mic23 for this protein. According to our results, Mic25/CHCHD6, Mic27/ApoOL and Mic23/ApoO appear to be periphery subunits of the MICOS complex, because their depletion does not affect cristae morphology or stability of other components.

Fig 1. Expression of FLAG-tagged components of the MIB and MICOS complexes.

(A-F) HeLa cells were grown on cover slips and transfected using Lipofectamine 2000 with pCDNA3 plasmids carrying information for FLAG-tagged Sam50 (A), Mic60/Mitofilin (B), Mic19/CHCHD3 (C), Mic25/CHCHD6 (D), Mic27/ApoOL (E) or Mic23/ApoO (F). Cells were then labeled with MitoTracker (red channel), fixed and immunostained stained using antibodies directed against the FLAG-tag (green channel) or Tom20 (A, blue channel) and corresponding, fluorophore-coupled secondary antibodes. In the upper right corner enlarged sections can be seen. Scale bar represents 10 μm.

Fig 2. Steady state levels of MIB and MICOS components in knockdown cell lines.

(A-F) Inducible knockdown cell lines, carrying shRNA that downregulates Sam50 (A, sam50kd-2), Mic60/Mitofilin (B, mflkd-2), Mic19/CHCHD3 (C, chchd3kd-2), Mic25/CHCHD6 (D, chchd6kd-3), Mic27/ApoOL (E, apoolkd-2) or Mic23/ApoO (F, apookd-4) were generated. Cells were induced with doxycycline (Dox) for 7 days and mitochondria were isolated from non-induced (-Dox) and induced cells (+Dox). 25 and 50 μg of mitochondrial protein from-Dox and +Dox samples were analyzed by SDS-PAGE and western blot, using antibodies directed against Sam50, Mic60/Mitofilin, Mic19/CHCHD3, Mic25/CHCHD6, Mic27/ApoOL, Mic23/ApoO, Mic10/MINOS1 and SDHA. SDHA—succinate dehydrogenase, a component of the mitochondrial respiratory complex II.

Fig 3. Mitochondrial morphology in knockdown cell lines.

(A-F) Cells from inducible knockdown cell lines, carrying shRNA that downregulates Sam50 (A, sam50kd-2), Mic60/Mitofilin (B, mflkd-2), Mic19/CHCHD3 (C, chchd3kd-2), Mic25/CHCHD6 (D, chchd6kd-3), Mic27/ApoOL (E, apoolkd-2) or Mic23/ApoO (F, apookd-4) were grown on coverslips and induced with doxycycline (Dox) for 7 days. After fixation, mitochondria were decorated with anti-Tom20 antibody and Cy5-coupled secondary antibody. Cells were analyzed by fluorescence microscopy. Enlarged sections of the shown pictures are represented in the upper right corner. Scale bar represents 10 μm. (G) Average circularity as a measurement of mitochondrial fragmentation was analyzed for 10 fields of view per sample (~200 cells) using Image J. pLV-THM, an empty vector cell line, and a mtx2kd-2 cell line carrying shRNA against Metaxin 2 served as controls (S2 Fig.). Significance was established using student’s t-test on the average circularity data. Data were normalized by setting the non-induced (-Dox) values to 1. * p<0.05, *** p<0.0001, n.s. not significant.

Fig 4. Transmission electron microscopy of MIB and MICOS components knockdown cell lines.

(A-F) Inducible knockdown cells as described in Fig. 2, carrying shRNA that downregulates Sam50 (A, sam50kd-2), Mic60/Mitofilin (B, mflkd-2), Mic19/CHCHD3 (C, chchd3kd-2), Mic25/CHCHD6 (D, chchd6kd-3), Mic27/ApoOL (E, apoolkd-2) or Mic23/ApoO (F, apookd-4) were seeded on cover slips, fixed and analyzed using transmission electron microscopy. All cells were induced with doxycycline (Dox) for 7 days, except chchd3kd-2, which was induced for 14 days. Scale bar represents 500 nm. The graphs on the right hand side represent mean values of at least hundred mitochondria counted from two different regions of two different samples ± SD. The mitochondria were divided into two groups, abnormal (Abn.) with circular stacks of inner membrane that appear instead of usual cristae and normal (Norm.) where the usual parallel cristae folds are visible.

Fig 5. Mic23/ApoO is co-precipitated with Mic60/Mitofilin and its import is affected by depletion of Mic60/Mitofilin and Sam50.

(A) Knockdown of Mic60/Mitofilin, Sam50 and Mic27/ApoOL was induced for 7 days using doxycycline (Dox) in the respective knockdown cell line (mflkd-2 for Mic60/Mitofilin, sam50kd-2 for Sam50 and apoolkd-2 for Mic27/ApoOL). Mitochondria were isolated and 50 μg of mitochondria per lane was incubated with ³⁵S-labeled Mic23/ApoO for indicated times. Lower panels represent western blot controls of the knockdown and loading, using antibodies against Mic60/Mitofilin, Sam50 or Mic27/ApoOL and Hsp60 or SDHA. Hsp60—heat shock protein 60, SDHA—succinate dehydrogenase, a component of the mitochondrial respiratory complex II. (B) Pulse-chase experiment was performed with 50 μg of HeLa mitochondria per lane, which were incubated with ³⁵S-labeled Mic23/ApoO for 20 min to allow formation of the Mic23/ApoO import complex. Mitochondria were reisolated, incubated in the import buffer for indicated times and analyzed by BN-PAGE and autoradiography. (C) Inducible knockdown cell line carrying shRNA that downregulates Sam50 (sam50kd-2) was induced for 7 days with doxycycline (Dox) and mitochondria were isolated from non-induced (-Dox) and induced cells (+Dox). 50 μg of mitochondrial protein per lane was solubilized in 1% digitonin buffer and analyzed by blue native (BN)-PAGE and western blot, using antibodies directed against Sam50, Mic60/Mitofilin or Mic19/CHCHD3, and, as control, SDHA (lower panels). SDHA—succinate dehydrogenase, a component of the mitochondrial respiratory complex II. Asterisks indicate three protein complexes observed after ³⁵S-labeled Mic23/ApoO import or western blot analysis of the steady state levels of Sam50, Mic60/Mitofilin and Mic19/CHCHD3. (D) Co-immunoprecipitation was performed by targeting Mic60/Mitofilin with specific antibodies and analyzing the precipitates by SDS-PAGE and western blot, using Mic60/Mitofilin, Mic23/ApoO and Tim44 antibodies. PBS was used as a negative control. Tim44—translocase of the inner mitochondrial membrane 44.