Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy.

The Rubicon-WIPI axis regulates exosome biogenesis during ageing.

Cells release intraluminal vesicles in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNA interference screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for intraluminal vesicle formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for age-dependent changes in exosome quantity and quality.

YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus.

The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the forming autophagosome. Using quantitative proteomic analysis and two novel Golgiphagy reporter systems, we found that the five-pass transmembrane Golgi-resident proteins YIPF3 and YIPF4 constitute a Golgiphagy receptor. The interaction of this complex with LC3B, GABARAP, and GABARAPL1 is dependent on a LIR motif within YIPF3 and putative phosphorylation sites immediately upstream; the stability of the complex is governed by YIPF4. Expression of a YIPF3 protein containing a mutated LIR motif caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays reported here may be readily adapted to different experimental contexts to help deepen our understanding of Golgiphagy.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence.

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB-HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria-lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage-induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence.

Lysophagy protects against propagation of α-synuclein aggregation through ruptured lysosomal vesicles.

The neuron-to-neuron propagation of misfolded α-synuclein (αSyn) aggregates is thought to be key to the pathogenesis of synucleinopathies. Recent studies have shown that extracellular αSyn aggregates taken up by the endosomal-lysosomal system can rupture the lysosomal vesicular membrane; however, it remains unclear whether lysosomal rupture leads to the transmission of αSyn aggregation. Here, we applied cell-based αSyn propagation models to show that ruptured lysosomes are the pathway through which exogenous αSyn aggregates transmit aggregation, and furthermore, this process was prevented by lysophagy, i.e., selective autophagy of damaged lysosomes. αSyn aggregates accumulated predominantly in lysosomes, causing their rupture, and seeded the aggregation of endogenous αSyn, initially around damaged lysosomes. Exogenous αSyn aggregates induced the accumulation of LC3 on lysosomes. This LC3 accumulation was not observed in cells in which a key regulator of autophagy, RB1CC1/FIP200, was knocked out and was confirmed as lysophagy by transmission electron microscopy. Importantly, RB1CC1/FIP200-deficient cells treated with αSyn aggregates had increased numbers of ruptured lysosomes and enhanced propagation of αSyn aggregation. Furthermore, various types of lysosomal damage induced using lysosomotropic reagents, depletion of lysosomal enzymes, or more toxic species of αSyn fibrils also exacerbated the propagation of αSyn aggregation, and impaired lysophagy and lysosomal membrane damage synergistically enhanced propagation. These results indicate that lysophagy prevents exogenous αSyn aggregates from escaping the endosomal-lysosomal system and transmitting aggregation to endogenous cytosolic αSyn via ruptured lysosomal vesicles. Our findings suggest that the progression and severity of synucleinopathies are associated with damage to lysosomal membranes and impaired lysophagy.