Autophagy has burgeoned rapidly as a field of study because of its evolutionary conservation, the diversity of intracellular cargoes degraded and recycled by this machinery, the mechanisms involved, as well as its physiological relevance to human health and disease. This self-eating process was initially viewed as a non-selective mechanism used by eukaryotic cells to degrade and recycle macromolecules in response to stress; we now know that various cellular constituents, as well as pathogens, can also undergo selective autophagy. In contrast to non-selective autophagy, selective autophagy pathways rely on a plethora of selective autophagy receptors (SARs) that recognize and direct intracellular protein aggregates, organelles and pathogens for specific degradation. Although SARs themselves are not highly conserved, their modes of action and the signalling cascades that activate and regulate them are. Recent yeast studies have provided novel mechanistic insights into selective autophagy pathways, revealing principles of how various cargoes can be marked and targeted for selective degradation.

The maintenance of intracellular NAD+/NADH homeostasis across multiple, subcellular compartments requires the presence of NADH-shuttling proteins, which circumvent the lack of permeability of organelle membranes to these cofactors. Very little is known regarding these proteins in the methylotrophic yeast, Pichia pastoris. During the study of the subcellular locations of these shuttling proteins, which often have dual subcellular locations, it became necessary to develop new ways to detect the weak peroxisomal locations of some of these proteins. We have developed a novel variation of the traditional Bimolecular Fluorescence Complementation (BiFC), called divergent BiFC, to detect intraorganellar colocalization of two noninteracting proteins based on their proximity-based protein crowding within a small subcellular compartment, rather than on the traditional protein-protein interactions expected for BiFC. This method is used to demonstrate the partially peroxisomal location of one such P. pastoris NADH-shuttling protein, malate dehydrogenase B, only when cells are grown in oleate, but not when grown in methanol or glucose. We discuss the mode of NADH shuttling in P. pastoris and the physiological basis of the medium-dependent compartmentalization of PpMdhB.

Organelle division and segregation are important in cellular homeostasis. Peroxisomes (POs) and mitochondria share a core division machinery and mechanism of membrane scission. The division of each organelle is interdependent not only on the other but also on other organelles, reflecting the dynamic communication between subcellular compartments, even as they coordinate the exchange of metabolites and signals. We highlight common and unique mechanisms involved in the fission of these organelles under the premise that much can be gleaned regarding the division of one organelle based on information available for the other.

Peroxisomes are conserved organelles of eukaryotic cells with important roles in cellular metabolism, human health, redox homeostasis, as well as intracellular metabolite transfer and signaling. We review here the current status of the different co-existing modes of biogenesis of peroxisomal membrane proteins demonstrating the fascinating adaptability in their targeting and sorting pathways. While earlier studies focused on peroxisomes as autonomous organelles, the necessity of the ER and potentially even mitochondria as sources of peroxisomal membrane proteins and lipids has come to light in recent years. Additionally, the intimate physical juxtaposition of peroxisomes with other organelles has transitioned from being viewed as random encounters to a growing appreciation of the expanding roles of such inter-organellar membrane contact sites in metabolic and regulatory functions. Peroxisomal quality control mechanisms have also come of age with a variety of mechanisms operating both during biogenesis and in the cellular response to environmental cues.

UNLABELLED: TRIM37 gene mutations cause mulibrey (muscle-liver-brain-eye) nanism, a severe growth disorder with prenatal onset. Although TRIM37 depletion normally induces apoptosis, patients with TRIM37 mutations have a high risk of developing tumors, suggesting that there may be an alternative pro-survival mechanism for TRIM37-deficient tumor cells. We find that TRIM37 interacts with MTOR and RRAGB proteins, enhances the MTOR-RRAGB interaction and promotes lysosomal localization of MTOR, thereby activating amino acid-stimulated MTORC1 signaling. In response to loss of TRIM37 functions, phosphorylation of TFEB is significantly reduced, resulting in its translocation into the nucleus enabling its transcriptional activation of genes involved in lysosome biogenesis and macroautophagy/autophagy. The enhanced autophagy depends on the inhibition of MTORC1 signaling and may serve as an alternative mechanism to survive the loss of TRIM37 functions. Our study unveils a positive role of TRIM37 in regulating the MTORC1-TFEB axis and provides mechanistic insights into the pathogenesis of mulibrey nanism, as well as potential therapeutic treatment. ABBREVIATIONS: ACTB: actin beta; ATG: autophagy related; CASP3: caspase3; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; CTS: cathepsin proteases; CTSL: cathepsin L; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LMNB1: lamin B1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; mulibrey: muscle-liver-brain-eye; NAC: N-acetyl-L-cysteine; PARP1: poly(ADP-ribose) polymerase 1; RAP2A: member of RAS oncogene family; RHEB: Ras homolog enriched in brain; ROS: reactive oxygen species; RPS6KB1: ribosomal protein S6 kinase B1; RRAGB: Ras related GTP binding B; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TRIM37: tripartite motif containing 37.

Peroxisomes are conserved organelles of eukaryotic cells with important roles in cellular metabolism, human health, redox homeostasis, as well as intracellular metabolite transfer and signaling. We review here the current status of the different co-existing modes of biogenesis of peroxisomal membrane proteins demonstrating the fascinating adaptability in their targeting and sorting pathways. While earlier studies focused on peroxisomes as autonomous organelles, the necessity of the ER and potentially even mitochondria as sources of peroxisomal membrane proteins and lipids has come to light in recent years. Additionally, the intimate physical juxtaposition of peroxisomes with other organelles has transitioned from being viewed as random encounters to a growing appreciation of the expanding roles of such inter-organellar membrane contact sites in metabolic and regulatory functions. Peroxisomal quality control mechanisms have also come of age with a variety of mechanisms operating both during biogenesis and in the cellular response to environmental cues.

Most proteins destined for the peroxisomal matrix depend on the peroxisomal targeting signals (PTSs), which require the PTS receptor PEX5, whose deficiency causes fatal human peroxisomal biogenesis disorders (PBDs). TRIM37 gene mutations cause muscle-liver-brain-eye (mulibrey) nanism. We found that TRIM37 localizes in peroxisomal membranes and ubiquitylates PEX5 at K464 by interacting with its C-terminal 51 amino acids (CT51), which is required for PTS protein import. PEX5 mutations (K464A or ΔCT51), or TRIM37 depletion or mutation, reduce PEX5 abundance by promoting its proteasomal degradation, thereby impairing its functions in cargo binding and PTS protein import in human cells. TRIM37 or PEX5 depletion induces apoptosis and enhances sensitivity to oxidative stress, underscoring the cellular requirement for functional peroxisomes. Therefore, TRIM37-mediated ubiquitylation stabilizes PEX5 and promotes peroxisomal matrix protein import, suggesting that mulibrey nanism is a new PBD.

TRIM37 gene mutations cause mulibrey (muscle-liver-brain-eye) nanism, a severe growth disorder with prenatal onset. Although TRIM37 depletion normally induces apoptosis, patients with TRIM37 mutations have a high risk of developing tumors, suggesting that there may be an alternative pro-survival mechanism for TRIM37-deficient tumor cells. We find that TRIM37 interacts with MTOR and RRAGB proteins, enhances the MTOR-RRAGB interaction and promotes lysosomal localization of MTOR, thereby activating amino acid-stimulated MTORC1 signaling. In response to loss of TRIM37 functions, phosphorylation of TFEB is significantly reduced, resulting in its translocation into the nucleus enabling its transcriptional activation of genes involved in lysosome biogenesis and macroautophagy/autophagy. The enhanced autophagy depends on the inhibition of MTORC1 signaling and may serve as an alternative mechanism to survive the loss of TRIM37 functions. Our study unveils a positive role of TRIM37 in regulating the MTORC1-TFEB axis and provides mechanistic insights into the pathogenesis of mulibrey nanism, as well as potential therapeutic treatment.

Abbreviations

ACTB: actin beta; ATG: autophagy related; CASP3: caspase3; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; CTS: cathepsin proteases; CTSL: cathepsin L; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LMNB1: lamin B1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; mulibrey: muscle-liver-brain-eye; NAC: N-acetyl-L-cysteine; PARP1: poly(ADP-ribose) polymerase 1; RAP2A: member of RAS oncogene family; RHEB: Ras homolog enriched in brain; ROS: reactive oxygen species; RPS6KB1: ribosomal protein S6 kinase B1; RRAGB: Ras related GTP binding B; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TRIM37: tripartite motif containing 37.

Increasingly, various 'omics data are contributing significantly to our understanding of novel biological processes, but it has not been possible to iteratively elucidate hierarchical functions in complex phenomena. We describe a general systems biology approach called Active Interaction Mapping (AI-MAP), which elucidates the hierarchy of functions for any biological process. Existing and new 'omics data sets can be iteratively added to create and improve hierarchical models which enhance our understanding of particular biological processes. The best datatypes to further improve an AI-MAP model are predicted computationally. We applied this approach to our understanding of general and selective autophagy, which are conserved in most eukaryotes, setting the stage for the broader application to other cellular processes of interest. In the particular application to autophagy-related processes, we uncovered and validated new autophagy and autophagy-related processes, expanded known autophagy processes with new components, integrated known non-autophagic processes with autophagy and predict other unexplored connections.