Article Figures & Data
Figures
- Figure 1. EMRE CAD faces the intermembrane space and mediates EMRE–MICU1 interaction.
(A) Tagging EMRE with a FLAG epitope tag at its C terminus does not impair its function. HEK293T cells expressing indicated proteins were permeabilized and mitochondrial Ca2+ uptake was measured by monitoring extramitochondrial Ca2+ clearance. Bar graph shows quantification of Ca2+ uptake rates and Western blot shows EMRE expression. (n = 4). ATP5A serves as loading control. (B) C-terminal FLAG tag does not impair EMRE–MCU and EMRE–MICU1 interactions. EMRE FLAG and control SDHB-FLAG were immunoprecipitated, and immunoprecipitates were blotted for MCU and MICU1. ATP5A serves as loading control. (C) Proteinase K treatment of isolated mitochondria in the presence of increased detergent concentration. EMRE-FLAG is degraded by proteinase K at the same detergent concentration as TIMM23, an inner mitochondrial membrane protein. (D) Mitochondria were isolated from WT or EMRE KO cells that stably express the indicated proteins. Mitoplasts (mitochondria without outer membranes) were prepared and treated with PEG5K-maleimide. A 5-kD mass addition to EMRE protein was detected by Western blotting. (D, E) Schematic shows EMRE membrane topology and the position of the amino acids that were mutated to cysteines for PEGylation experiments shown in (D). EMRE aa 64–85 were predicted to form its transmembrane domain using TMHMM (Sonnhammer et al, 1998). (F) EMRE DDD domain is not required for mitochondrial calcium uptake. Mitochondrial calcium uptake rates of WT and EMRE KO cells stably expressing the indicated proteins (n = 4). (G) Charge-conserving mutations of the six aspartic acids of EMRE to glutamic acid restores EMRE–MICU1 interaction. WT EMRE–FLAG or EMRE–FLAG with the indicated mutations were stably expressed in EMRE KO cells, immunoprecipitated, and immunoprecipitates were subjected to Western blotting to detect EMRE–MICU1 interaction. Data information: In (A, F), data are presented as mean ± SD.
- Figure S1. EMRE S53C, S64C, and 108C are functional proteins.
Mutation of EMRE S53 and S64C to cysteine or addition of a cysteine at the C terminus of EMRE does not impair its function. HEK293T cells expressing indicated proteins were permeabilized and mitochondrial Ca2+ uptake was measured by monitoring extramitochondrial Ca2+ clearance. Bar graph shows quantification of Ca2+ uptake rates relative to WT HEK293T cells (n = 4). Data information: data are presented as mean ± SD.
- Figure 2. Carboxyl-terminal helices of Dictyostelium discoideum MCU (DdMCU) confer EMRE-independent Ca2+ uptake to Homo sapiens MCU (HsMCU).
(A) Schematic shows helices and sheets of HsMCU and DdMCU as predicted by PSIPRED. Two transmembrane domains are labeled. (B, C) Schematic summarizes the domain structure of HsMCU, DdMCU, and the chimeric proteins. FLAG-tagged proteins were stably expressed in MCU KO and EMRE KO HEK293T cells. Mitochondrial Ca2+ uptake rates in control WT and chimera expressing cells were measured and normalized to those of WT cells (n = 3–4). Expression of chimeras was detected by Western blotting using anti-FLAG antibody. TOM20 serves as loading control. Data information: in (B, C), data are presented as mean ± SD.
- Figure 3. EMRE dependence domain of Homo sapiens MCU is a 10- amino acid–long region located C-terminal to TM2.
(A, B) Schematic summarizes the domain structure of chimeric proteins. FLAG-tagged proteins were stably expressed in mitochondrial calcium uniporter (MCU) KO and EMRE KO HEK293T cells. Mitochondrial Ca2+ uptake rates in control WT and chimera expressing cells were measured and normalized to those of WT cells (n = 3–4). (C) Alignment of MCU protein from indicated species was done using CLUSTALW and amino acids were color-coded using BoxShade. Black boxes show identical amino acids, gray boxes show similar amino acids. TM1, TM2 and EMRE dependence domain are indicated. Data information: In (A, B), data are presented as mean ± SD.
- Figure S2. Homo sapiens MCU–Dictyostelium discoideum MCU (HsMCU–DdMCU) chimera break points.
Arrowheads point to the conserved amino acids of HsMCU and DdMCU that were chosen as break points in chimeras. Alignment of HsMCU and DdMCU was done using CLUSTALW, and amino acids were color coded using BoxShade. Black boxes show identical amino acids and gray boxes show similar amino acids.
- Figure S3. Comparison of Homo sapiens MCU–Dictyostelium discoideum MCU chimera protein expression and function.
(A) mitochondrial calcium uniporter (MCU) KO or EMRE KO cells that stably express the indicated FLAG-tagged proteins were lysed, and lysates were analyzed by Western blotting using anti-FLAG antibody. ATP5A serves as loading control. (B) MCU KO and EMRE KO cells were infected with virus as described in material and methods to generate 1× infected cells after puromycin selection. The same cells were infected again with twice the amount of original virus to produce 3× infected cells. Mitochondrial Ca2+ uptake rates of control WT, 1× infected and 3× infected cells were measured. Bar graph shows mitochondrial Ca2+ uptake rates relative to WT control cells (n = 3). Western blot shows expression levels of FLAG-tagged H. sapiens MCU and chimera 5. ATP5A serves as loading control. (B) Data information: in (B), data are presented as mean ± SD.
- Figure S4. Expression of Homo sapiens MCU, Dictyostelium discoideum MCU, or chimeras do not alter mitochondrial membrane potential.
Control WT, MCU KO, or EMRE KO cells expressing the indicated proteins were permeabilized and incubated with TMRM. TMRM fluorescence was measured in the absence and presence of uncdupler CCCP. All cells showed comparable mitochondrial membrane potential (n = 3). Data information: Data are presented as mean ± SD.
- Figure S5. Representative mitochondrial Ca2+ uptake traces.
WT, mitochondrial calcium uniporter (MCU) KO, or EMRE KO HEK293T cell expressing the indicated proteins were permeabilized and mitochondrial Ca2+ uptake was measured by monitoring extramitochondrial Ca2+ clearance. The arrows indicate addition of Ca2+. Each graph shows data from WT and MCU KO or EMRE KO cells, in addition to data from cell lines that express the indicated chimeras.
- Figure 4. EMRE directly interacts with TM1 of mitochondrial calcium uniporter (MCU).
(A) MCU TM and C-terminal helices are required for EMRE–MCU interaction. Untagged EMRE and indicated FLAG-tagged MCU proteins were co-expressed in MCU KO HEK293T cells by transient transfection, FLAG-tagged proteins were immunoprecipitated and immunoprecipitates were analyzed for the presence of EMRE by Western blotting. ATP5A serves as loading control. EMRE–MCU interaction was evident both in immunoprecipitates and in lysates through stabilization of EMRE. (B) Schematic summarizes EMRE-chimera binding data and highlights the importance of MCU TM and C-terminal helices for EMR–EMCU interaction. (C, D) EMRE–Homo sapiens MCU cysteine cross-linking experiments show direct binding of MCU TM1 residues A241 and A251 to EMRE F77 and T82, respectively. (C, D) H. sapiens MCU that contains only one cysteine at amino acid 241 (C) or 251 (D) were stably co-expressed with indicated EMRE proteins. WT EMRE does not contain any cysteines and served as a control. Mitochondria were isolated from cells, and cysteine–cysteine cross-linking was induced using copper phenanthroline. MCU-FLAG was immunoprecipitated and the presence of an ∼40 kD cross-linked EMRE-MCU band was detected under non-reducing conditions by Western blotting. Lysates were prepared in parallel under reducing conditions and were blotted to detect indicated proteins. ATP5A serves as loading control. Numbers indicate the locations of molecular weight standards.
- Figure S6. Functional characterization of mitochondrial calcium uniporter (MCU) and EMRE proteins used for cysteine cross-linking experiments.
(A, B, C) MCU KO HEK293T cells expressing indicated proteins were permeabilized and mitochondrial Ca2+ uptake was measured by monitoring extramitochondrial Ca2+ clearance. Bar graphs show Ca2+ uptake rates relative to control WT cells (n = 3–4). (A, B, C) MCU–EMRE cross-linking in TM1 of MCU (A), in TM2 or MCU (B), in EMRE-dependence domain of MCU (C) are shown. Data information: data are presented as mean ± SD.
- Figure 5. EMRE directly interacts with TM2 and EMRE dependence domain (EDD) of mitochondrial calcium uniporter (MCU).
(A, B) EMRE–Homo sapiens MCU (HsMCU) cysteine cross-linking experiment shows direct binding of MCU TM2 residues I270 and M276C to EMRE I84 and P76, respectively. (A, B) HsMCU that contains only one cysteine at amino acid 270 (A) 276 (B) was stably co-expressed with indicated EMRE proteins in MCU KO cells. WT EMRE does not contain any cysteines and served as a control. Mitochondria were isolated from cells and cysteine–cysteine cross-linking was induced using copper phenanthroline. MCU-FLAG was immunoprecipitated and the presence of an ∼40 kD cross-linked EMRE–MCU band was detected under non-reducing conditions by Western blotting using EMRE antibody. Lysates were prepared in parallel under reducing conditions and were blotted to detect indicated proteins. ATP5A serves as loading control. Numbers indicate the locations of molecular weight standards. (C) EMRE–HsMCU cysteine cross-linking experiments show direct binding of MCU EDD residues E293 and D296 to EMRE K59 and K62. (A) Cross-linking and sample processing were performed as in (A). (D) Schematic showing MCU and EMRE amino acids that directly interact with each other in the membrane and in the matrix. EDD is shown in red. (E) Schematic showing HsMCU, HsMCU with Caenorhabditis elegans MCU EDD, Hs EMRE, and HsEMRE with C. elegans EMRE N-terminal domain. (F, G) These constructs were used in (F, G). (F) Mitochondrial Ca2+ uptake rates in control WT or EMRE KO cells stably expressing HsEMRECeNterm together with HsMCU or HsMCUCeEDD were measured and normalized to those of WT cells (n = 3–4). MCU forms a functional channel only if its EDD interacts with EMRE. (G) MCU–FLAG was immunoprecipitated from EMRE KO cells that stably express HsMCU or HsMCUCeEDD with HsEMRE or HsEMRECeNterm after DSP-mediated cross-linking. Immunoprecipitates and lysates were analyzed with Western blotting for the presence of indicated proteins. An interaction with MCU and EMRE was observed only if EDD and EMRE originated from the same species. (H) Model shows the proposed mechanism of EMRE function in the uniporter. In the absence of EMRE, Ca2+ ions cannot exit the channel because of blockage of the pore by EDD. Binding of EMRE leads to a conformational change in EDD and allows exit of Ca2+ ions into the matrix. (F) Data information: in (F), data are presented as mean ± SD.
- Figure S7. The region homologous to EMRE dependence domain (EDD) appears flexible in fungi.
High-resolution structures of mitochondrial calcium uniporter (MCU) tetramers from four different fungal species are shown. The region that is homologous to EDD or amino acids that surround the EDD in these structures are shown in red. Dotted blue lines show amino acids that are omitted in these structures, all of which overlap with EDD. In the Metarhizium acridum MCU structure, one MCU chain shows alpha helical EDD, whereas the same region in the neighboring chain is not structured. Protein Data Bank IDs of these structures are as follows: M. acridum (6C5W); N. crassa (5KUJ); N. fischeri (6D7W); C. europaea (6DNF).
- Figure S8. Alignment of human and Caenorhabditis elegans mitochondrial calcium uniporter (MCU) TM1, TM2, and EMRE dependence domain, and EMRE N-terminal domain.
Alignments of MCU or EMRE from human and C. elegans were done using CLUSTALW, and amino acids were color coded using BoxShade. Black boxes show identical amino acids and gray boxes show similar amino acids. Purple box indicates EMRE dependence domain. Arrowheads indicate Homo sapiens MCU and HsEMRE amino acids that cross-link.
Supplementary Materials
Supplemental Data 1.
Annotated DNA and corresponsing protein sequences of constructs used.[LSA-2020-00718_Supplemental_Data_1.docx]
