Jiefei Geng

The Atg8 and Atg12 ubiquitin‐like conjugation systems in macroautophagy

As a lysosomal/vacuolar degradative pathway that is conserved in eukaryotic organisms, autophagy mediates the turnover of long‐lived proteins and excess or aberrant organelles. The main characteristic of autophagy is the formation of a double‐membrane vesicle, the autophagosome, which envelops part of the cytoplasm and delivers it to the lysosome/vacuole for breakdown and eventual recycling of the degradation products. Among the approximately 30 autophagy‐related (Atg) genes identified so far, there are two ubiquitin‐like proteins, Atg12 and Atg8. Analogous to ubiquitination, Atg12 is conjugated to Atg5 by Atg7—an E1‐like protein—and Atg10—an E2‐like protein. Similarly, Atg7 and Atg3 are the respective E1‐like and E2‐like proteins that mediate the conjugation of Atg8 to phosphatidylethanolamine. Both Atg12–Atg5 and Atg8 localize to the developing autophagosome. The Atg12–Atg5 conjugate facilitates the lipidation of Atg8 and directs its correct subcellular localization. Atg8–phosphatidylethanolamine is probably a scaffold protein that supports membrane expansion and the amount present correlates with the size of autophagosomes.

SNARE proteins are required for macroautophagy

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.

A Genomic Screen for Yeast Mutants Defective in Selective Mitochondria Autophagy

Mitophagy is the process of selective mitochondrial degradation via autophagy, which has an important role in mitochondrial quality control. Very little is known, however, about the molecular mechanism of mitophagy. A genome-wide yeast mutant screen for mitophagy-defective strains identified 32 mutants with a block in mitophagy, in addition to the known autophagy-related (ATG) gene mutants. We further characterized one of these mutants, ylr356wDelta that corresponds to a gene whose function has not been identified. YLR356W is a mitophagy-specific gene that was not required for other types of selective autophagy or macroautophagy. The deletion of YLR356W partially inhibited mitophagy during starvation, whereas there was an almost complete inhibition at post-log phase. Accordingly, we have named this gene ATG33. The new mutants identified in this analysis will provide a useful foundation for researchers interested in the study of mitochondrial homeostasis and quality control.

Post-Golgi Sec Proteins Are Required for Autophagy in Saccharomyces cerevisiae

Macroautophagy is linked to various diseases in humans, including cancer and neurodegeneration. The morphological hallmark is the formation of the double-membrane autophagosome, which is the most complex aspect of macroautophagy. We demonstrate a role for post-Golgi Sec proteins, Sec2 and Sec4, in autophagosome formation.

Arp2 Links Autophagic Machinery with the Actin Cytoskeleton

Macroautophagy involves lysosomal/vacuolar elimination of long-lived proteins and entire organelles from the cytosol. The process begins with formation of a double-membrane vesicle that sequesters bulk cytoplasm, or a specific cargo destined for lysosomal/vacuolar delivery. The completed vesicle fuses with the lysosome/vacuole limiting membrane, releasing its content into the organelle lumen for subsequent degradation and recycling of the resulting macromolecules. A majority of the autophagy-related (Atg) proteins are required at the step of vesicle formation. The integral membrane protein Atg9 cycles between certain intracellular compartments and the vesicle nucleation site, presumably to supply membranes necessary for macroautophagic vesicle formation. In this study we have tracked the movement of Atg9 over time in living cells by using real-time fluorescence microscopy. Our results reveal that an actin-related protein, Arp2, briefly colocalizes with Atg9 and directly regulates the dynamics of Atg9 movement. We propose that proteins of the Arp2/3 complex regulate Atg9 transport for specific types of autophagy.

Quantitative analysis of autophagy-related protein stoichiometry by fluorescence microscopy

In yeast, ∼31 autophagy-related (Atg) proteins have been identified. Most of them reside at the phagophore assembly site (PAS), although the function of the PAS mostly remains unclear. One reason for the latter is the lack of stoichiometric information regarding the Atg proteins at this site. We report the application of fluorescence microscopy to study the amount of Atg proteins at the PAS. We find that an increase in the amount of Atg11 at the PAS enhances the recruitment of Atg8 and Atg9 to this site and facilitates the formation of more cytoplasm-to-vacuole targeting vesicles. In response to autophagy induction, the amount of most Atg proteins remains unchanged at the PAS, whereas we see an enhanced recruitment of Atg8 and 9 at this site. During autophagy, the amount of Atg8 at the PAS showed a periodic change, indicating the formation of autophagosomes. Application of this method and further analysis will provide more insight into the functions of Atg proteins.

RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS

Axonal pathology and necroptosis in ALS Necroptosis, a non–caspase-dependent form of cell death, can be reduced in disease states by inhibiting a kinase called RIPK1. Until now, no human mutations have been linked to necroptosis. Ito et al. show that loss of optineurin, which is encoded by a gene that has been implicated in the human neurodegenerative disorder ALS (amyotrophic lateral sclerosis), results in sensitivity to necroptosis and axonal degeneration. When RIPK1-kinase dependent signaling is disrupted in mice that lack optineurin, necroptosis is inhibited and axonal pathology is reversed. Science, this issue p. 603 Inflammatory and cell death mechanisms underlie axonal pathology in amyotrophic lateral sclerosis. Mutations in the optineurin (OPTN) gene have been implicated in both familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of this protein in the central nervous system (CNS) and how it may contribute to ALS pathology are unclear. Here, we found that optineurin actively suppressed receptor-interacting kinase 1 (RIPK1)–dependent signaling by regulating its turnover. Loss of OPTN led to progressive dysmyelination and axonal degeneration through engagement of necroptotic machinery in the CNS, including RIPK1, RIPK3, and mixed lineage kinase domain–like protein (MLKL). Furthermore, RIPK1- and RIPK3-mediated axonal pathology was commonly observed in SOD1G93A transgenic mice and pathological samples from human ALS patients. Thus, RIPK1 and RIPK3 play a critical role in mediating progressive axonal degeneration. Furthermore, inhibiting RIPK1 kinase may provide an axonal protective strategy for the treatment of ALS and other human degenerative diseases characterized by axonal degeneration.

Positive or Negative Roles of Different Cyclin-Dependent Kinase Pho85-Cyclin Complexes Orchestrate Induction of Autophagy in Saccharomyces cerevisiae

As a major intracellular degradation pathway, autophagy is tightly regulated to prevent cellular dysfunction in all eukaryotic cells. The rapamycin-sensitive Tor kinase complex 1 is a major regulator of autophagy. Several other nutrient-sensory kinases also play critical roles to precisely modulate autophagy; however, the network of regulatory mechanisms remains largely elusive. We used genetic analyses to elucidate the mechanism by which the stress-responsive, cyclin-dependent kinase Pho85 and its corresponding cyclin complexes antagonistically modulate autophagy in Saccharomyces cerevisiae. When complexed with cyclins Pho80 and Pcl5, Pho85 negatively regulates autophagy through downregulating the protein kinase Rim15 and the transcription factors Pho4 and Gcn4. The cyclins Clg1, Pcl1, and Pho80, in concert with Pho85, positively regulate autophagy through promoting the degradation of Sic1, a negative regulator of autophagy that targets Rim15. Our results suggest a model in which Pho85 and its cyclin complexes have opposing roles in autophagy regulation.

The Golgi as a potential membrane source for autophagy

In macroautophagy (hereafter autophagy), a morphological hallmark is the formation of double-membrane vesicles called autophagosomes that sequester and deliver cytoplasmic components to the lysosome/vacuole for degradation. This process begins with an initial sequestering compartment, the phagophore, which expands into the mature autophagosome. A tremendous amount of work has been carried out to elucidate the mechanism of how the autophagosome is formed. However, an important missing piece in this puzzle is where the membrane comes from. Independent lines of evidence have shown that pre-existing organelles may continuously supply lipids to support autophagosome formation. In our analysis, we identified several components of the late stage secretory pathway that may redirect Golgi-derived membrane to autophagosome formation in response to starvation conditions.

Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death.

Metabolic stress caused by perturbation of receptor tyrosine kinase FLT3 sensitizes cancer cells to autophagy inhibition and leads to excessive activation of chaperone-mediated autophagy, which triggers metabolic catastrophe in cancer cells through the degradation of HK2.