Rab32 uses its effector reticulon 3L to trigger autophagic degradation of mitochondria-associated membrane (MAM) proteins.

BACKGROUND: Rab32 is a small GTPase associated with multiple organelles but is particularly enriched at the endoplasmic reticulum (ER). Here, it controls targeting to mitochondria-ER contacts (MERCs), thus influencing composition of the mitochondria-associated membrane (MAM). Moreover, Rab32 regulates mitochondrial membrane dynamics via its effector dynamin-related protein 1 (Drp1). Rab32 has also been reported to induce autophagy, an essential pathway targeting intracellular components for their degradation. However, no autophagy-specific effectors have been identified for Rab32. Similarly, the identity of the intracellular membrane targeted by this small GTPase and the type of autophagy it induces are not known yet. RESULTS: To investigate the target of autophagic degradation mediated by Rab32, we tested a large panel of organellar proteins. We found that a subset of MERC proteins, including the thioredoxin-related transmembrane protein TMX1, are specifically targeted for degradation in a Rab32-dependent manner. We also identified the long isoform of reticulon-3 (RTN3L), a known ER-phagy receptor, as a Rab32 effector. CONCLUSIONS: Rab32 promotes degradation of mitochondrial-proximal ER membranes through autophagy with the help of RTN3L. We propose to call this type of selective autophagy "MAM-phagy".

Expression of a T39N mutant Rab32 protein arrests mitochondria movement within neurites of differentiated SH-SY5Y cells.

We have shown that multiple sclerosis (MS) and endoplasmic reticulum (ER) stress induce Rab32, an ER/mitochondria-localized small GTPase. High levels of both dominant-active (Q85L) or dominant-inactive (T39N) Rab32 are toxic to neurons. While Rab32Q85L interacts with its effector Drp1 to promote mitochondria fission, it is unclear how Rab32T39N could result as toxic to neurons. Given the perinuclear clustering of mitochondria observed upon transfection of inactive Rab32, we hypothesized Rab32T39N could stall mitochondria within neurites. The movement of mitochondria depends on kinesin-binding Miro proteins. High cytosolic [Ca2+] is bound by an EF hand motif within Miro proteins, resulting in mitochondrial arrest. Consistent with increased cytosolic [Ca2+], expression of Rab32T39N arrests mitochondria movement within neurites.

Plastic mitochondria-endoplasmic reticulum (ER) contacts use chaperones and tethers to mould their structure and signaling.

Mitochondria-endoplasmic reticulum (ER) contacts (MERCs), biochemically isolated as the mitochondria-associated membrane (MAM), were discovered on electron micrographs in the early 1950s. Since the 1990s, we know that the two organelles exchange lipids and Ca2+ ions at these membrane contacts. Already in the very first publication on this intracellular structure, the extreme plasticity of the structure was obvious. Recent progress has now confirmed that ER and mitochondria move closer to deepen physical contacts under conditions of ER stress, hypoxia, or short-term nutrient deprivation, while nutrient over-supply is one situation that lessens contacts. Signaling associated with these intracellular events moulds the contact site ultrastructure, in particular during autophagy, apoptosis and alterations of mitochondria metabolism. Tethering complexes, as well as key MAM proteins including chaperones of the ER and mitochondriacontrol the plasticity of MERC structures. It has become evident that altered MAM composition and changes in MAM plasticity are critical factors for the development of cancer, neurodegeneration and the metabolic syndrome.

Over Six Decades of Discovery and Characterization of the Architecture at Mitochondria-Associated Membranes (MAMs).

The discovery of proteins regulating ER-mitochondria tethering including phosphofurin acidic cluster sorting protein 2 (PACS-2) and mitofusin-2 has pushed contact sites between the endoplasmic reticulum (ER) and mitochondria into the spotlight of cell biology. While the field is developing rapidly and controversies have come and gone multiple times during its history, it is sometimes overlooked that significant research has been done decades ago with the original discovery of these structures in the 1950s and the first characterization of their function (and coining of the term mitochondria-associated membrane, MAM) in 1990. Today, an ever-increasing array of proteins localize to the MAM fraction of the endoplasmic reticulum (ER) to regulate the interaction of this organelle with mitochondria. These mitochondria-ER contacts, sometimes referred to as MERCs, regulate a multitude of biological functions, including lipid metabolism, Ca2+ signaling, bioenergetics, inflammation, autophagy, mitochondrial structure, and apoptosis.

Cancer: Untethering Mitochondria from the Endoplasmic Reticulum?

Following the discovery of the mitochondria-associated membrane (MAM) as a hub for lipid metabolism in 1990 and its description as one of the first examples for membrane contact sites at the turn of the century, the past decade has seen the emergence of this structure as a potential regulator of cancer growth and metabolism. The mechanistic basis for this hypothesis is that the MAM accommodates flux of Ca2+ from the endoplasmic reticulum (ER) to mitochondria. This flux then determines mitochondrial ATP production, known to be low in many tumors as part of the Warburg effect. However, low mitochondrial Ca2+ flux also reduces the propensity of tumor cells to undergo apoptosis, another cancer hallmark. Numerous regulators of this flux have been recently identified as MAM proteins. Not surprisingly, many fall into the groups of tumor suppressors and oncogenes. Given the important role that the MAM could play in cancer, it is expected that proteins mediating its formation are particularly implicated in tumorigenesis. Examples for such proteins are mitofusin-2 and phosphofurin acidic cluster sorting protein 2 that likely act as tumor suppressors. This review discusses how these proteins that mediate or regulate ER-mitochondria tethering are (or are not) promoting or inhibiting tumorigenesis. The emerging picture of MAMs in cancer seems to indicate that in addition to the downregulation of mitochondrial Ca2+ import, MAM defects are but one way how cancer cells control mitochondria metabolism and apoptosis.

Of yeast, mice and men: MAMs come in two flavors.

The past decade has seen dramatic progress in our understanding of membrane contact sites (MCS). Important examples of these are endoplasmic reticulum (ER)-mitochondria contact sites. ER-mitochondria contacts have originally been discovered in mammalian tissue, where they have been designated as mitochondria-associated membranes (MAMs). It is also in this model system, where the first critical MAM proteins have been identified, including MAM tethering regulators such as phospho-furin acidic cluster sorting protein 2 (PACS-2) and mitofusin-2. However, the past decade has seen the discovery of the MAM also in the powerful yeast model system Saccharomyces cerevisiae. This has led to the discovery of novel MAM tethers such as the yeast ER-mitochondria encounter structure (ERMES), absent in the mammalian system, but whose regulators Gem1 and Lam6 are conserved. While MAMs, sometimes referred to as mitochondria-ER contacts (MERCs), regulate lipid metabolism, Ca2+ signaling, bioenergetics, inflammation, autophagy and apoptosis, not all of these functions exist in both systems or operate differently. This biological difference has led to puzzling discrepancies on findings obtained in yeast or mammalian cells at the moment. Our review aims to shed some light onto mechanistic differences between yeast and mammalian MAM and their underlying causes. REVIEWERS: This article was reviewed by Paola Pizzo (nominated by Luca Pellegrini), Maya Schuldiner and György Szabadkai (nominated by Luca Pellegrini).

TMX1 determines cancer cell metabolism as a thiol-based modulator of ER-mitochondria Ca2+ flux.

The flux of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria regulates mitochondria metabolism. Within tumor tissue, mitochondria metabolism is frequently repressed, leading to chemotherapy resistance and increased growth of the tumor mass. Therefore, altered ER-mitochondria Ca(2+) flux could be a cancer hallmark, but only a few regulatory proteins of this mechanism are currently known. One candidate is the redox-sensitive oxidoreductase TMX1 that is enriched on the mitochondria-associated membrane (MAM), the site of ER-mitochondria Ca(2+) flux. Our findings demonstrate that cancer cells with low TMX1 exhibit increased ER Ca(2+), accelerated cytosolic Ca(2+) clearance, and reduced Ca(2+) transfer to mitochondria. Thus, low levels of TMX1 reduce ER-mitochondria contacts, shift bioenergetics away from mitochondria, and accelerate tumor growth. For its role in intracellular ER-mitochondria Ca(2+) flux, TMX1 requires its thioredoxin motif and palmitoylation to target to the MAM. As a thiol-based tumor suppressor, TMX1 increases mitochondrial ATP production and apoptosis progression.