Systematic In‐Depth Proteomic Analysis of Mitochondria‐Associated Endoplasmic Reticulum Membranes in Mouse and Human Testes

Abstract

Mitochondria-associated endoplasmic reticulum membranes (MAMs) regulate important cellular functions including calcium signaling, bioenergetics, and apoptosis during neurodevelopment and carcinogenesis, but its function in male reproduction and spermatogenesis remains enigmatic because the field lacks a complete understanding of the proteome within testis MAMs. To better understand the biological processes and molecular functions of MAM in testes, a global mass spectrometry-based proteomic evaluation of MAM proteins from human and mouse testes are reported here, respectively. The evaluation and analysis showed that the components of MAM were highly conserved not only between different species (human and mouse) but also between different tissues (testes and brains). Bioinformatics interrogation of these MAM protein catalogues uncovered that 815 new potential linkages specifically existed in mouse testes compared with mouse brains. In addition, a comparative analysis showed that 1347 proteins (account for ≈96.56%) were highly conservatively expressed in both human and mouse testis MAMs. Furthermore, functional analysis revealed that testis-specific MAM proteins were related to spermatogenesis, male gamete generation, as well as sexual reproduction. The data identified, for the first time, numerous MAM proteins in mouse and human testes, which provide a possibility to define the relationship between testis MAM proteins and reproductive diseases.

Mitofusin2 cooperates with Nuage-associated proteins and involves mRNA translational machinery in controlling mRNA fates during spermatogenesis

“…When the full length of MFN2 was ectopically co-expressed with MIWI, TDRKH, and DDX4 in HEK293T cells, MFN2 was pulled down by MIWI, TDRKH, and DDX4, indicating MFN2’s ability to interact with MIWI, TDRKH and DDX4 proteins (Figure.4F-G). Importantly, our previous studies have shown that all of these interacting proteins are within the MAM structures of mouse and human testes (Wang et al, 2018), suggesting MFN2 may function with the Nuage-associated proteins in maintaining MAM structure integrity.…”

“…Alteration of MAMs increasingly was reported in several human diseases, highlighted by neurodegenerative diseases(Paillusson et al, 2016). Our previous work has demonstrated an abundance of MAMs in mouse and human testes and identified a large portion of MAM proteins in testes (Wang et al, 2018), which suggests that MAM proteins may have critical functions in spermatogenesis.…”

“…Mitofusins (MFN1/2), the homologs of Fzo in yeast and Drosophila, are the critical regulators of mitochondrial fusion in mammalian cells and enriched in MAMs (Hales and Fuller, 1997; Santel and Fuller, 2001; Wang et al, 2018). Loss of either Mfn1 or Mfn2 in mice leads to embryonic lethality, and cultured cells obtained from the mutant mouse embryos display overtly fragmented mitochondria (Chen et al, 2003).…”

Split-TurboID enables contact-dependent proximity labeling in cells

“…We compared our split-TurboID proteomes to previous ER-mitochondria contact proteomes obtained by other methods. The four comparison datasets were: (1) a MAM preparation from human tissue (35), (2) a study combining MAM isolation and proximity labeling (36), (3) our previous study using APEX labeling on the OMM and ERM separately, followed by dataset intersection (9), and (4) the Contact-ID-generated ER-mitochondria proteome (21). Figure 5B shows that specificity, as measured by the fraction of proteins with prior OMM or ERM annotation, is similar for all of the proximity labeling-based studies, but much poorer for the MAM dataset, which contains contaminants from many other organelles.…”

“…Known ERM and OMM proteins (as annotated by GOCC) are labeled blue and red, respectively; ERM and OMM dual-annotated proteins are colored purple, and all other proteins are shown in black. Cutoffs used to filter the mass (35). (C) Markov clustering of split-TurboID proteome using protein-protein interaction scores from the STRING database (33).…”