The MICOS component Mic60 displays a conserved membrane-bending activity that is necessary for normal cristae morphology

Abstract

The inner membrane (IM) of mitochondria displays an intricate, highly folded architecture and can be divided into two domains: the inner boundary membrane adjacent to the outer membrane and invaginations toward the matrix, called cristae. Both domains are connected by narrow, tubular membrane segments called cristae junctions (CJs). The formation and maintenance of CJs is of vital importance for the organization of the mitochondrial IM and for mitochondrial and cellular physiology. The multisubunit mitochondrial contact site and cristae organizing system (MICOS) was found to be a major factor in CJ formation. In this study, we show that the MICOS core component Mic60 actively bends membranes and, when inserted into prokaryotic membranes, induces the formation of cristae-like plasma membrane invaginations. The intermembrane space domain of Mic60 has a lipid-binding capacity and induces membrane curvature even in the absence of the transmembrane helix. Mic60 homologues from α-proteobacteria display the same membrane deforming activity and are able to partially overcome the deletion of Mic60 in eukaryotic cells. Our results show that membrane bending by Mic60 is an ancient mechanism, important for cristae formation, and had already evolved before α-proteobacteria developed into mitochondria.

Figure 1.

Purification and reconstitution of S. cerevisiae Mic60. (A) Domain architecture of Mic60 S. cerevisiae. (B) Expression and purification of recombinant Mic60 analyzed by SDS-PAGE with subsequent Coomassie Brilliant Blue staining. (C) Circular dichroism spectra of Mic60 in urea and renatured in DDM. mdeg, milidegree of molar elliptisity. (D) Flotation assay of empty LUVs and Mic60-containing vesicles as indicated, separated in a noncontinuous Histodenz density gradient. (E) Fractionation of the Histodenz density gradient centrifugation of Mic60-containing vesicles analyzed by SDS-PAGE Coomassie Brilliant Blue staining. (F) Sodium carbonate extraction assay of Mic60-containing LUVs analyzed by SDS-PAGE Coomassie Brilliant Blue staining. (G) Flotation assay of LUVs containing Tim50 and the epsin1 ENTH domain as indicated, separated in a noncontinuous Histodenz gradient. (H) Fractionation of floated samples from G analyzed by SDS-PAGE Coomassie Brilliant Blue staining. (I) Sodium carbonate extraction assay of Tim50- and ENTH-containing LUVs analyzed by SDS-PAGE Coomassie Brilliant Blue staining. P, pellet; S, supernatant.

Figure 2.

Mic60 induces high degrees of curvature in model membranes. (A) Electron micrographs of negatively stained LUVs with the indicated additives. (B) Dynamic light scattering analysis of LUV size distribution. Error bars represent SEM. (C) GUVs in the absence and presence of the indicated proteins. GUVs were labeled with rhodamine-PE, and proteins were labeled with Alexa Fluor 488. (A and C) Bars: (A) 100 nm; (C) 10 µm. (D) Histogram of GUV morphologies in the absence and presence of various proteins. For each condition, a minimum of 100 GUVs from three preparations were counted, and the values given in the histogram are absolute numbers. ILVs, intraluminal vesicles; ILSs, intraluminal sheets.

Figure 3.

Mic60 induces membrane invaginations in prokaryotic membranes. (A) Electron micrograph of an E. coli cell. (B) Electron micrograph of an E. coli cell expressing MBP-Mic60. Arrows mark Mic60-induced intracytoplasmaic membranes. The zoomed image shows that membranes invaginate from the plasma membrane. (C) Electron micrographs of immunogold-labeled E. coli cells (anti-Mic60). (D) Cell fractionation of E. coli cells expressing MBP-Mic60 analyzed by Western blot. Anti-MBP antibody was used. (E) Quantification of the immunogold labeling. The histogram represents RD. RD was evaluated by counting the number of specific gold particles (1,481) in 27 E. coli cells (n = 27). Error bars represent SD. (F) Electron micrographs of E. coli cells expressing the indicated proteins. (G) Electron micrographs of E. coli cells expressing the IMS domain of Mic60 as an MBP fusion protein. The zoomed image shows internal membrane structures connected to the plasma membrane. The right image shows an immunogold-labeled E. coli cell. (H) EM image of LUVs in the presence of Mic60^IMS. Bars, 100 nm.

Figure 4.

α-Proteobacterial Mic60 homologues deform the membrane in vitro and in vivo and rescue depletion of Mic60 in eukaryotic cells. (A) Electron micrographs of E. coli cells expressing Mic60 from P. denitrificans as an MBP fusion protein. (B) Cell fractionation of E. coli cells expressing Mic60^P.d. analyzed by Western blot. Anti-MBP antibody was used. (C) Electron micrographs of E. coli cells expressing Mic60 from R. sphaeroides as an MBP fusion protein. (A and C) Arrows mark intracytoplasmaic membranes. Zoomed images show internal membrane structures connected to the plasma membrane. (D) Cell fractionation of E. coli cells expressing Mic60^R.s. analyzed by Western blot. Anti-MBP antibody was used. (E) Electron micrographs of negatively stained LUVs in the absence or presence of the indicated proteins. (F) Fractionation of floated Mic60^P.d.- and Mic60^R.s.-containing LUVs analyzed by SDS-PAGE Coomassie Brilliant Blue staining. (G) Representative fluorescent microscopy images (top) and EM images (bottom) of yeast cells with the indicated genetic backgrounds and mitochondrial ultrastructures. (A, C, E, and G) Bars: (A, C, and E) 100 nm; (G, top) 2.5 µm; (G, bottom) 300 nm. (H) Quantitative evaluation of the mitochondrial IM ultrastructure as detected in cell sections. Mitochondria with at least one clear connection to the inner boundary membrane (see wild-type [wt] EM image) were counted as unchanged. Those with stacked or onion-shaped cristae membranes (see mic60Δ EM image) and without connections to the boundary membrane were counted as changed. The given values are absolute numbers provided by the counting of mitochondria (number of mitochondria as indicated in the figure) with different structural features.