Regulated interaction of ID2 with the anaphase-promoting complex links progression through mitosis with reactivation of cell-type-specific transcription.

Tissue-specific transcriptional activity is silenced in mitotic cells but it remains unclear whether the mitotic regulatory machinery interacts with tissue-specific transcriptional programs. We show that such cross-talk involves the controlled interaction between core subunits of the anaphase-promoting complex (APC) and the ID2 substrate. The N-terminus of ID2 is independently and structurally compatible with a pocket composed of core APC/C subunits that may optimally orient ID2 onto the APCCDH1 complex. Phosphorylation of serine-5 by CDK1 prevented the association of ID2 with core APC, impaired ubiquitylation and stabilized ID2 protein at the mitosis-G1 transition leading to inhibition of basic Helix-Loop-Helix (bHLH)-mediated transcription. The serine-5 phospho-mimetic mutant of ID2 that inefficiently bound core APC remained stable during mitosis, delayed exit from mitosis and reloading of bHLH transcription factors on chromatin. It also locked cells into a "mitotic stem cell" transcriptional state resembling the pluripotent program of embryonic stem cells. The substrates of APCCDH1 SKP2 and Cyclin B1 share with ID2 the phosphorylation-dependent, D-box-independent interaction with core APC. These results reveal a new layer of control of the mechanism by which substrates are recognized by APC.

A pair of transporters controls mitochondrial Zn2+ levels to maintain mitochondrial homeostasis.

Zn2+ is required for the activity of many mitochondrial proteins, which regulate mitochondrial dynamics, apoptosis and mitophagy. However, it is not understood how the proper mitochondrial Zn2+ level is achieved to maintain mitochondrial homeostasis. Using Caenorhabditis elegans, we reveal here that a pair of mitochondrion-localized transporters controls the mitochondrial level of Zn2+. We demonstrate that SLC-30A9/ZnT9 is a mitochondrial Zn2+ exporter. Loss of SLC-30A9 leads to mitochondrial Zn2+ accumulation, which damages mitochondria, impairs animal development and shortens the life span. We further identify SLC-25A25/SCaMC-2 as an important regulator of mitochondrial Zn2+ import. Loss of SLC-25A25 suppresses the abnormal mitochondrial Zn2+ accumulation and defective mitochondrial structure and functions caused by loss of SLC-30A9. Moreover, we reveal that the endoplasmic reticulum contains the Zn2+ pool from which mitochondrial Zn2+ is imported. These findings establish the molecular basis for controlling the correct mitochondrial Zn2+ levels for normal mitochondrial structure and functions.

Defective arginine metabolism impairs mitochondrial homeostasis in Caenorhabditiselegans.

Arginine catabolism involves enzyme-dependent reactions in both mitochondria and the cytosol, defects in which may lead to hyperargininemia, a devastating developmental disorder. It is largely unknown if defective arginine catabolism has any effects on mitochondria. Here we report that normal arginine catabolism is essential for mitochondrial homeostasis in Caenorhabditiselegans. Mutations of the arginase gene argn-1 lead to abnormal mitochondrial enlargement and reduced adenosine triphosphate (ATP) production in C. elegans hypodermal cells. ARGN-1 localizes to mitochondria and its loss causes arginine accumulation, which disrupts mitochondrial dynamics. Heterologous expression of human ARG1 or ARG2 rescued the mitochondrial defects of argn-1 mutants. Importantly, genetic inactivation of the mitochondrial basic amino acid transporter SLC-25A29 or the mitochondrial glutamate transporter SLC-25A18.1 fully suppressed the mitochondrial defects caused by argn-1 mutations. These findings suggest that mitochondrial damage probably contributes to the pathogenesis of hyperargininemia and provide clues for developing therapeutic treatments for hyperargininemia.

The amino acid transporter SLC-36.1 cooperates with PtdIns3P 5-kinase to control phagocytic lysosome reformation.

Phagocytic removal of apoptotic cells involves formation, maturation, and digestion of cell corpse-containing phagosomes. The retrieval of lysosomal components following phagolysosomal digestion of cell corpses remains poorly understood. Here we reveal that the amino acid transporter SLC-36.1 is essential for lysosome reformation during cell corpse clearance in Caenorhabditis elegans embryos. Loss of slc-36.1 leads to formation of phagolysosomal vacuoles arising from cell corpse-containing phagosomes. In the absence of slc-36.1, phagosome maturation is not affected, but the retrieval of lysosomal components is inhibited. Moreover, loss of PPK-3, the C. elegans homologue of the PtdIns3P 5-kinase PIKfyve, similarly causes accumulation of phagolysosomal vacuoles that are defective in phagocytic lysosome reformation. SLC-36.1 and PPK-3 function in the same genetic pathway, and they directly interact with one another. In addition, loss of slc-36.1 and ppk-3 causes strong defects in autophagic lysosome reformation in adult animals. Our findings thus suggest that the PPK-3-SLC-36.1 axis plays a central role in both phagocytic and autophagic lysosome formation.

The lysine catabolite saccharopine impairs development by disrupting mitochondrial homeostasis.

Amino acid catabolism is frequently executed in mitochondria; however, it is largely unknown how aberrant amino acid metabolism affects mitochondria. Here we report the requirement for mitochondrial saccharopine degradation in mitochondrial homeostasis and animal development. In Caenorhbditis elegans, mutations in the saccharopine dehydrogenase (SDH) domain of the bi-functional enzyme α-aminoadipic semialdehyde synthase AASS-1 greatly elevate the lysine catabolic intermediate saccharopine, which causes mitochondrial damage by disrupting mitochondrial dynamics, leading to reduced adult animal growth. In mice, failure of mitochondrial saccharopine oxidation causes lethal mitochondrial damage in the liver, leading to postnatal developmental retardation and death. Importantly, genetic inactivation of genes that raise the mitochondrial saccharopine precursors lysine and α-ketoglutarate strongly suppresses SDH mutation-induced saccharopine accumulation and mitochondrial abnormalities in C. elegans Thus, adequate saccharopine catabolism is essential for mitochondrial homeostasis. Our study provides mechanistic and therapeutic insights for understanding and treating hyperlysinemia II (saccharopinuria), an aminoacidopathy with severe developmental defects.

C. elegans-based screen identifies lysosome-damaging alkaloids that induce STAT3-dependent lysosomal cell death.

Lysosomes are degradation and signaling centers within the cell, and their dysfunction impairs a wide variety of cellular processes. To understand the cellular effect of lysosome damage, we screened natural small-molecule compounds that induce lysosomal abnormality using Caenorhabditis elegans (C. elegans) as a model system. A group of vobasinyl-ibogan type bisindole alkaloids (ervachinines A-D) were identified that caused lysosome enlargement in C. elegans macrophage-like cells. Intriguingly, these compounds triggered cell death in the germ line independently of the canonical apoptosis pathway. In mammalian cells, ervachinines A-D induced lysosomal enlargement and damage, leading to leakage of cathepsin proteases, inhibition of autophagosome degradation and necrotic cell death. Further analysis revealed that this ervachinine-induced lysosome damage and lysosomal cell death depended on STAT3 signaling, but not RIP1 or RIP3 signaling. These findings suggest that lysosome-damaging compounds are promising reagents for dissecting signaling mechanisms underlying lysosome homeostasis and lysosome-related human disorders.

GOP-1 promotes apoptotic cell degradation by activating the small GTPase Rab2 in C. elegans.

Apoptotic cells generated by programmed cell death are engulfed by phagocytes and enclosed within plasma membrane-derived phagosomes. Maturation of phagosomes involves a series of membrane-remodeling events that are governed by the sequential actions of Rab GTPases and lead to formation of phagolysosomes, where cell corpses are degraded. Here we identified gop-1 as a novel regulator of apoptotic cell clearance in Caenorhabditis elegans Loss of gop-1 affects phagosome maturation through the RAB-5-positive stage, causing defects in phagosome acidification and phagolysosome formation, phenotypes identical to and unaffected by loss of unc-108, the C. elegans Rab2 GOP-1 transiently associates with cell corpse-containing phagosomes, and loss of its function abrogates phagosomal association of UNC-108. GOP-1 interacts with GDP-bound and nucleotide-free UNC-108/Rab2, disrupts GDI-UNC-108 complexes, and promotes activation and membrane recruitment of UNC-108/Rab2 in vitro. Loss of gop-1 also abolishes association of UNC-108 with endosomes, causing defects in endosome and dense core vesicle maturation. Thus, GOP-1 is an activator of UNC-108/Rab2 in multiple processes.

The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy.

Autophagy-dependent clearance of ubiquitinated and aggregated proteins is critical to protein quality control, but the underlying mechanisms are not well understood. Here, we report the essential role of the BEACH (beige and Chediak-Higashi) and WD40 repeat-containing protein WDR81 in eliminating ubiquitinated proteins through autophagy. WDR81 associates with ubiquitin (Ub)-positive protein foci, and its loss causes accumulation of Ub proteins and the autophagy cargo receptor p62. WDR81 interacts with p62, facilitating recognition of Ub proteins by p62. Furthermore, WDR81 interacts with LC3C through canonical LC3-interacting regions in the BEACH domain, promoting LC3C recruitment to ubiquitinated proteins. Inactivation of LC3C or defective autophagy results in accumulation of Ub protein aggregates enriched for WDR81. In mice, WDR81 inactivation causes accumulation of p62 bodies in cortical and striatal neurons in the brain. These data suggest that WDR81 coordinates p62 and LC3C to facilitate autophagic removal of Ub proteins, and provide important insights into CAMRQ2 syndrome, a WDR81-related developmental disorder.

The lysosomal cathepsin protease CPL-1 plays a leading role in phagosomal degradation of apoptotic cells in Caenorhabditis elegans.

During programmed cell death, the clearance of apoptotic cells is achieved by their phagocytosis and delivery to lysosomes for destruction in engulfing cells. However, the role of lysosomal proteases in cell corpse destruction is not understood. Here we report the identification of the lysosomal cathepsin CPL-1 as an indispensable protease for apoptotic cell removal in Caenorhabditis elegans. We find that loss of cpl-1 function leads to strong accumulation of germ cell corpses, which results from a failure in degradation rather than engulfment. CPL-1 is expressed in a variety of cell types, including engulfment cells, and its mutation does not affect the maturation of cell corpse-containing phagosomes, including phagosomal recruitment of maturation effectors and phagosome acidification. Of importance, we find that phagosomal recruitment and incorporation of CPL-1 occurs before digestion of cell corpses, which depends on factors required for phagolysosome formation. Using RNA interference, we further examine the role of other candidate lysosomal proteases in cell corpse clearance but find that they do not obviously affect this process. Collectively, these findings establish CPL-1 as the leading lysosomal protease required for elimination of apoptotic cells in C. elegans.