Identification of cold-inducible mitochondrial proteins in brown adipose tissue
To identify candidate genes involved in the thermogenic function of BAT mitochondria, we analyzed publicly available gene expression data for genes that are enriched in BAT, as compared to epididymal WAT (eWAT) (GSE92844, (Mo et al., 2017)), and induced by cold stimulus (GSE70437, (Marcher et al., 2015)) (Fig. S1A). From this analysis, we identified a cluster of 144 genes that were both enriched in BAT and induced by cold stimulation (Fig. S1A). Among these 144 genes, we identified a subgroup of 31 genes which were annotated as mitochondrial genes including Ucp1 (Fig. S1B, Table S1).
Mitochondrial gene transcript levels and protein levels are frequently discordant, so we performed a complementary analysis with proteomics data (Forner et al., 2009). By analyzing the intersection of proteins that are BAT-enriched proteins and also induced by cold, 26 mitochondrial proteins were identified (Fig. S1C, Table S1). After combining the results of the transcriptomic and proteomic analyses, three candidates, Ucp1, Letmd1, and Acsl5 were identified (Fig. S1D). Ucp1 is a well-established mitochondrial protein required for BAT thermogenesis and Acsl5 has also been identified as an important regulator of whole-body energy metabolism in previous studies (Bowman et al., 2016). However, in contrast to these two genes, the role of Letmd1 in BAT has not been explored.
Letmd1 is a BAT-enriched protein that is upregulated in response to thermogenic demand
To begin to understand the role of Letmd1 in BAT, we examined Letmd1 expression during brown adipocyte differentiation in an immortalized brown preadipocyte (iBPA) cell culture model (Uldry M. et al., 2006). During iBPA differentiation into brown adipocytes, Letmd1 expression increased at both RNA and protein levels (Figs. 1A and 1B). In adult mice, Letmd1 protein was highly enriched in BAT relative to WAT and other tissues, including mitochondria-rich muscle, indicating that tissue mitochondrial content is not a determinant of Letmd1 expression levels (Fig. 1C). Thermogenic demand is known to increase during the early postnatal period of mice and we found that Letmd1 transcript and protein levels increased during this period (Figs. 1D and 1E).
We next compared Letmd1 expression in BAT from mice housed at thermoneutrality (30℃), room temperature (23℃), or cold (6℃) conditions and observed an inverse relationship between BAT Letmd1 expression and ambient temperature (Figs. 1F and 1G). Notably, when cold-exposed mice were re-acclimated to room temperature (Figs. 1H and 1I), cold-induced expression of Letmd1 reverted to room temperature levels (Fig. 1J), a pattern also seen for Ucp1 expression (Fig. 1K). Taken together, Letmd1 is enriched in mature brown adipocytes and shows dynamic regulation as a function of external cues determining the physiological demand for BAT thermogenesis.
Letmd1 is an integral membrane protein that targets to the mitochondrial matrix
Next, we investigated the subcellular localization of LETMD1 protein in brown adipocytes. Sequence analysis with MitoFates (Fukasawa et al., 2015) identified a putative mitochondrial targeting sequence (MTS) (1-28aa) and a cleavage site by mitochondrial processing peptidases (MPP) (Fig. 2A). In agreement with sequence analysis, immunofluorescence imaging of LETMD1 overexpressing iBPA cells displayed a mitochondrial pattern of localization (Fig. 2B). In addition to a mitochondrial presequence, LETMD1 protein contains two putative transmembrane domains (82-97aa and 139-161aa) as predicted by TMHMM analysis (Krogh et al., 2001) (Fig. 2C).
To gain more information on the domain structure and cellular localization of Letmd1 protein, we analyzed the activity of a series of APEX2 fusion expression constructs. APEX2 is an engineered peroxidase whose subcellular localization is visualized by electron microscopy (EM) of staining patterns generated by peroxidase activity (Martell et al., 2012). High resolution details offered by EM analysis of APEX2 staining patterns has been successful for determining mitochondrial compartment-specific localization and protein domain topology (Rhee et al., 2013, Hung et al., 2014, Hung et al., 2017, Lee et al., 2017) (Fig. 2D). EM imaging of HEK293 cells transfected with Letmd1-APEX2 (C-terminus APEX fusion) and APEX2-Letmd1 (N-terminus fusion) expression constructs both showed matrix staining patterns indicating that the N-terminus and C-terminus of Letmd1 are exposed to the matrix (Fig. 2E). While we observed a typical mitochondrial matrix pattern of peroxidase staining in cells expressing MTS-APEX2, APEX2 with an N-terminus mitochondrial targeting sequence (MTS), cells expressing the Letmd1-APEX2 construct lacking the predicted MTS (1-28aa) abolished mitochondrial localization demonstrating functionality of the Letmd1 MTS (Fig. 2E).
In another approach, we utilized the proximity labeling activity of a mitochondrial matrix localized APEX2, Matrix-APEX2, to test whether Letmd1 contains a matrix localized domain. To this end, iBPA cells were transfected with Matrix-APEX2 and Letmd1 expressing vectors. Letmd1 protein was detected in the biotin-labeled fraction of cell lysates indicating matrix localization of Letmd1 protein (Fig. 2F). These results were further confirmed in vivo as endogenous Letmd1 protein was detected in the labeled fraction of BAT lysates from Matrix-APEX2 transgenic mice which harbor a Matrix-APEX2 transgene under the control of a general promoter (Park et al., 2021) (Fig. 2G). These results demonstrate that Letmd1 is a mitochondrial matrix protein that contains a functional N-terminal MTS and two transmembrane domains spanning the inner mitochondrial membrane (Fig. 2H).
Letmd1 is required for adaptive thermogenesis
To study the in vivo function of Letmd1, we generated Letmd1 knockout (KO) mice that are homozygous for a Letmd1 null allele. Compared to control mice, Letmd1 KO mice had increased body weight (Fig. 3A) in the absence of significant changes in food intake or physical activity (Figs. 3B and 3C). Gross examination of Letmd1 KO BAT tissue revealed a striking “whitened” appearance and histological analysis revealed dramatically enlarged lipid droplets in Letmd1 KO BAT compared to littermate controls (Fig. 3D). Furthermore, the expression of Ucp1 and thermogenic genes were markedly reduced in Letmd1 KO BAT from adult mice (Figs. 3E and 3F). This reduction in Ucp1 protein levels in Letmd1 KO BAT was observed immediately after birth and persists through the perinatal period when thermogenic demand first arises (Fig. 3G). These findings led us to test whether thermogenic function of BAT is altered in Letmd1 KO mice. Indeed, we found Letmd1 KO mice failed to maintain core body temperature during a cold challenge indicating a defect in adaptive thermogenesis (Fig. 3H). Infrared imaging of Letmd1 KO mice further confirmed the inability of Letmd1 KO mice to activate BAT and maintain body temperature during a cold challenge (Fig. 3I).
To further characterize physiological alterations in Letmd1 KO mice, we performed indirect calorimetry of control and Letmd1 KO mice during a cold challenge. At 25 ℃, we did not observe any difference in oxygen consumption (VO2) and carbon dioxide production (VCO2) between the groups. However, while both wild-type and Letmd1 KO mice rapidly increased VO2 and VCO2 upon initiation of a cold challenge, Letmd1 KO mice failed to maintain elevated levels of VO2 and VCO2 which is necessary for a sustained thermogenic response (Figs. 3J and 3K). This impaired response to cold stress in Letmd1 KO mice was accompanied by a failure to upregulate Ucp1 protein in BAT tissue (Fig. 3L). These results demonstrate an obligate in vivo requirement for Letmd1 function in the adaptive thermogenic response of BAT upon a cold challenge.
Letmd1 plays an essential role in maintaining BAT mitochondrial structure and respiratory function
To investigate the molecular role of Letmd1 in brown adipocyte function and adaptive thermogenesis, we performed RNA-seq analysis of Letmd1 KO and wild-type BAT. Gene ontology (GO) analysis of transcriptome changes revealed down-regulation of a broad array of genes involved in processes that occur in the mitochondrial matrix compartment in Letmd1 KO BAT (Fig. 4A). At the ultrastructural level, Letmd1 KO BAT contained dysmorphic mitochondria with sparse distended cristae, a principal site of OXPHOS complex (Fig. 4B). These data led us to directly examine OXPHOS complex components and function in Letmd1 KO BAT. At embryonic day 16.5, when thermogenic demand is absent, BAT OXPHOS complex proteins were unaffected by Letmd1 deficiency (Fig. 4C). However, during the early postnatal period, control BAT upregulated OXPHOS complex proteins in response to thermogenic demand but Letmd1 KO BAT failed to upregulate OXPHOS proteins during this critical period for BAT thermogenesis (Fig. 4D). The defective OXPHOS protein expression in Letmd1 KO BAT was further exacerbated in adult stages and showed a near complete loss of complex I and IV proteins (Fig. 4E).
To examine the functional consequence of OXPHOS complex deficiency in Letmd1 KO BAT, we measured respiratory function in scrambled control (SCR) and Letmd1 knockdown brown adipocytes (shLetmd1) in the presence or absence of isoproterenol stimulation. In the unstimulated state, we did not observe any significant differences in cellular respiration between SCR and shLetmd1 brown adipocytes (Figs. 4F and 4G). However, upon isoproterenol stimulation, we observed a dramatic reduction in maximal respiration of shLetmd1 cells, which is in sharp contrast with control (SCR) cells (Figs. 4F and 4G). Collectively, these data demonstrate a crucial role for Letmd1 in supporting brown adipocyte OXPHOS function in a stimulation-dependent manner.