LETMD1 regulates mitochondrial protein synthesis and import to guard brown fat mitochondrial integrity and function

Abstract

Thermogenic brown adipocytes (BAs) catabolize lipids to generate heat, representing powerful agents against the growing global obesity epidemic. We and others reported recently that LETMD1 is a BA-specific protein essential for mitochondrial structure and function, but the mechanisms of action remain unclear. We performed sequential digestion to demonstrate that LETMD1 is a trans-inner mitochondrial membrane protein. We then generated UCP1Cre-driven BA-specific Letmd1 knockout (Letmd1 ^UKO ) mice to show that Letmd1 ^UKO leads to protein aggregation, reactive oxidative stress, hyperpolarization, and mitophagy in BAs. We further employed TurboID proximity labeling to identify LETMD1-interacting proteins. Many candidate proteins are associated with mitochondrial ribosomes, protein import machinery, and electron transport chain complexes (ETC-I and ETC-IV). Using quantitative proteomics, we confirmed the elevated aggregations of ETC and mitochondrial ribosomal proteins, impairing mitochondrial protein synthesis in the Letmd1 ^UKO BAs. Therefore, LETMD1 may function to maintain mitochondrial proteostasis through regulating import of nuclear-encoded proteins and local protein translation in brown fat mitochondria.

Keywords: Cell; Cell biology; Cellular physiology; Molecular biology.

Graphical abstract

Figure 1

LETMD1 is a trans-IMM protein with a cell autonomous role in BAT (A) Mitochondrial swelling assay reveals LETMD1 localization on the IMM and matrix-facing portion. The addition of Isotonic buffer and Proteinase K disrupts OMM (Mic19 and CPT2 controls). The addition of Hypotonic buffer and Proteinase K causes swelling of IMM (MRP-L12 control). Hypotonic solution, proteinase K and Triton X-100 burst mitochondria. (B) Schematic representation of LETMD1 localization on IMM. (C) Schematic diagram of the Cre-LoxP recombination design used to generate knockout of Letmd1 (Letmd1^UKO) in UCP1+ cells. (D and E) Bodyweight (D) and adipose tissue weights (E) from two-month-old Ctrl and Letmd1^UKO mice (n = 5). (F) qPCR analysis of Letmd1 and Ucp1 expression in two-month-old CT and Letmd1^UKO BAT (n = 4). (G) Representative images of adipose tissues (F) from two-month-old Ctrl and Letmd1^UKO mice (n = 5). (H) Representative BAT images and H&E staining of Ctrl and Letmd1^UKO BAT at different developmental stages, including postnatal day 0 (P0), postnatal day 3 (P3), and postnatal day 21 (P21) (n = 5/stage). Scale bar: 100 μm. (I) H&E staining of adipose tissues from P60 (two-month-old) Ctrl and Letmd1^UKO mice. Scale bar: 100 μm. Data are presented as mean ± SEM. Two-tailed Student’s t test, ∗p ≤ 0.05, ∗∗p ≤ 0.01. Illustrations were created with Biorender.com.

Figure 2

LETMD1^UKO mice are cold intolerant and exhibit severe mitochondrial defects (A) Average rectal temperature of two-month-old Ctrl and Letmd1^UKO mice during acute cold exposure without food (n = 4). (B) iBAT temperature of Ctrl and Letmd1^UKO mice upon chronic cold exposure (n = 5). (C and D) Bodyweight (C) and adipose tissue weights (D) of Ctrl and Letmd1^UKO mice upon chronic cold exposure (n = 5). (E) Representative images of adipose tissue from two-month-old Ctrl and Letmd1^UKO mice at RT and after chronic cold exposure. (F) H&E staining of adipose tissue from Ctrl and Letmd1^UKO mice after chronic cold exposure. Scale bar: 100 μm. (G and H) Representative transmission electron microscopy (TEM) images of Ctrl and Letmd1^UKO BAT at RT (G) and after chronic cold exposure for 7 days (H). Scale bar for RT and chronic cold TEM lefthand images: 5 μm, righthand images: 1 μm. Red arrows in TEM images indicate swollen mitochondria morphology. Blue arrows indicate donut-shaped mitochondria morphology. (I and J) Quantification of the mitochondria count per field on TEM image and the number of cristae per mitochondrion (I) and quantification of mitochondria morphology type per field on TEM image (J) in Ctrl and Letmd1^UKO BAT at room temperature. Eight mitochondria from two pairs of mice were analyzed. (K) Relative mitochondrial DNA copy number in Ctrl and Letmd1^UKO BAT at room temperature (n = 4). (L) Quantification of mitochondria area in Ctrl and Letmd1^UKO BAT. Fifty mitochondria from two pairs of mice were quantified. Data are presented as mean ± SEM. Two-tailed Student’s t test, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 3

Letmd1^UKO BAT mitochondria have an altered proteome and exhibit autophagic stress (A) Schematic showing mitochondria isolation for mass spectrometry. (B) Volcano plot of differentially expressed proteins for Ctrl and Letmd1^UKO BAT mitochondria (n = 3). Each dot represents one protein. Red dots indicate upregulated protein expression in Letmd1^UKO BAT mitochondria. Blue dots indicate downregulated protein expression in Letmd1^UKO BAT mitochondria. Log₂ Difference threshold = 1.5. p value threshold = 0.01. (C) Heatmap of mitochondrial protein expression pattern in Ctrl and Letmd1^UKO BAT mitochondria (n = 3). (D) Schematic showing the process of mitophagy, involving the engulfment and degradation of damaged mitochondria. (E) Representative TEM images of Letmd1^UKO BAT mitochondria undergoing mitophagy events, including engulfment (i), lysosome fusion (ii) and degradation (iii), in comparison to Ctrl BAT mitochondria. Red arrows indicate the site of mitophagy. Scale bar: 1 μm. (F) qPCR analysis of autophagy markers Lc3b and Lamp1 in Ctrl and Letmd1^UKO BAT (n = 4). Data are presented as mean ± SEM. Two-tailed Student’s t test, ∗p ≤ 0.05, ∗∗p ≤ 0.01. Illustrations created with Biorender.com.

Figure 4

Letmd1^UKO mature brown adipocytes exhibit mitochondrial stress (A) Immunofluorescence staining of mature brown adipocytes with ROS. Quantification of ROS intensity reveals a significant increase in Letmd1^UKO mature brown adipocytes (n = 30 cells quantified/group). (B) Immunofluorescence staining of mature brown adipocytes with TMRE for mitochondrial membrane potential. Quantification of TMRE intensity reveals a significant increase in Letmd1^UKO mature brown adipocytes, indicative of hyperpolarization (n = 25 cells quantified/group). (C) Immunofluorescence staining of mature brown adipocytes with Fluo4 for intracellular calcium. Quantification of Fluo4 intensity reveals a significant decrease in Letmd1^UKO mature brown adipocytes (n = 32 cells quantified/group). Scale bar: 1 μm. Data are presented as mean ± SEM. Two-tailed Student’s t test, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 5

TurboID proximity labeling reveals a potential role of LETMD1 in mitochondrial import (A) Schematic of TurboID proximity labeling. Biotin-AMP can covalently label proteins proximal to LETMD1, up to ∼ 20nm. Schematic adapted from Cho et al., 2020. (B) LETMD1-TurboID fusion protein construct cloned into the pSBtet-Pur plasmid (Addgene #60507), harboring doxycycline-induced expression. The mitochondrial targeting sequence (MTS) of LETMD1 ensures mitochondria localization. The amino acid sequence GGGGSGGG was inserted between LETMD1 and TurboID as a linker. A Myc tag is included for detection in immunoblot and immunofluorescence analysis. (C) TurboID proximity labeling experimental design for mass spectrometry analysis. (D) Volcano plot resulting from LETMD1-TurboID proteomic analysis (n = 3). Red dots indicate enriched proteins, using Log2 Difference threshold = 1.5 and p value threshold = 0.01. (E–G) Tables of enriched proteins of interest, related to ETC complex I and complex IV assembly and function (E), mitochondrial protein import (F) and mitochondrial ribosome assembly and function (G). (H) List of all other enriched proteins, not of interest, with various functions. (I) Gene ontology (GO) molecular function analysis of enriched proteins. (J) Immunoblot analysis showing decreased expression of LETMD1 and electron transport chain complexes CI (NDUFB8), CIV (MT-CO1), and CIII (UQCRC2) in Letmd1^UKO BAT. (K) Gene ontology (GO) biological process analysis of enriched proteins. Illustrations created with Biorender.com.

Figure 6

Letmd1^UKO BAT exhibits protein aggregates but increased solubilization of complex I proteins (A) Representative TEM images of aggregate formation within Letmd1^UKO BAT mitochondria, compared to Ctrl BAT mitochondria. Red arrows indicate aggregates. Quantification reveals significantly increased protein aggregate formation within Letmd1^UKO BAT mitochondria. Twenty-two mitochondria from two pairs of mice were quantified. Scale bar: 5 μm. (B) Schematic showing the experimental design for isolation of cytosolic and mitochondrial fractions from Ctrl and Letmd1^UKO mice. Illustration created with Biorender.com. (C) Quantification of soluble and insoluble protein quantity from Ctrl and Letmd1^UKO cytosolic fractions (n = 3). (D) Quantification of soluble and insoluble protein quantity from Ctrl and Letmd1^UKO mitochondrial fractions (n = 3). (E) Heatmap of Letmd1^UKO BAT whole-cell soluble and insoluble proteins (n = 3) (p value cutoff <0.01). (F and G) Gene ontology (GO) analysis for molecular function (F) and biological process (G) of proteins that met significance threshold (p value < 0.01) in Letmd1^UKO aggregates. Data are presented as mean ± SEM. Two-tailed Student’s t test, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 7

Validation and illustration of LETMD1 association with respiratory chain complexes and mitochondrial protein (A) Two-dimensional analysis of oxidative phosphorylation complexes and LETMD1. Isolated BAT mitochondria were first separated by blue native electrophoresis (BN-PAGE, horizontal line) followed by denaturing (SDS-PAGE, vertical line). (B) Immunoblot analysis and quantification of nuclear and mitochondrial-encoded proteins reveals decreased expression of specific respiratory chain proteins. (C) Summary diagram showing the proposed role of LETMD1 in ribosomal and ETC protein import, mitochondrial protein synthesis and ETC complex assembly on the inner mitochondrial membrane. Illustration created with Biorender.com.