Fulvio Reggiori

Emerging regulation and functions of autophagy

Autophagy maintains cell, tissue and organism homeostasis through degradation. Complex post-translational modulation of the Atg (autophagy-related) proteins adds additional entry points for crosstalk with other cellular processes and helps define cell-type-specific regulations of autophagy. Beyond the simplistic view of a process exclusively dedicated to the turnover of cellular components, recent data have uncovered unexpected functions for autophagy and the autophagy-related genes, such as regulation of metabolism, membrane transport and modulation of host defenses — indicating the novel frontiers lying ahead.

Autophagy in the eukaryotic cell

The major cellular pathways for protein and organelle turnover are autophagy and proteasome-mediated degradation. These processes are important to maintain a well-controlled balance between anabolism and catabolism in order to have normal cell growth and development. They play an essential role

The Atg1-Atg13 complex regulates Atg9 and Atg23 retrieval transport from the pre-autophagosomal structure

To survive extreme environmental conditions, and in response to certain developmental and pathological situations, eukaryotic organisms employ the catabolic process of autophagy. Structures targeted for destruction are enwrapped by double-membrane vesicles, then delivered into the interior of the lysosome/vacuole. Despite the identification of many specific components, the molecular mechanism that directs formation of the sequestering vesicles remains largely unknown. We analyzed the trafficking of Atg23 and the integral membrane protein Atg9 in the yeast Saccharomyces cerevisiae. These components localize both to the pre-autophagosomal structure (PAS) and other cytosolic punctate compartments. We show that Atg9 and Atg23 cycle through the PAS in a process governed by the Atg1-Atg13 signaling complex. Atg1 kinase activity is essential only for retrograde transport of Atg23, while recycling of Atg9 requires additional factors including Atg18 and Atg2. We postulate that Atg9 employs a recycling system mechanistically similar to that used at yeast early and late endosomes.

Sorting of proteins into multivesicular bodies: ubiquitin‐dependent and ‐independent targeting

Yeast endosomes, like those in animal cells, invaginate their membranes to form internal vesicles. The resulting multivesicular bodies fuse with the vacuole, the lysosome equivalent, delivering the internal vesicles for degradation. We have partially purified internal vesicles and analysed their content. Besides the known component carboxypeptidase S (Cps1p), we identified a polyphosphatase (Phm5p), a presumptive haem oxygenase (Ylr205p/Hmx1p) and a protein of unknown function (Yjl151p/Sna3p). All are membrane proteins, and appear to be cargo molecules rather than part of the vesicle‐forming machinery. We show that both Phm5p and Cps1p are ubiquitylated, and that in a doa4 mutant, which has reduced levels of free ubiquitin, Cps1p, Phm5p and Hmx1p are mis‐sorted to the vacuolar membrane. Mutation of Lys 6 in the cytoplasmic tail of Phm5p disrupts its sorting, but sorting is restored, even in doa4 cells, by the biosynthetic addition of a single ubiquitin chain. In contrast, Sna3p enters internal vesicles in a ubiquitin‐independent manner. Thus, ubiquitin acts as a signal for the partitioning of some, but not all, membrane proteins into invaginating endosomal vesicles.

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.

An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis

Despite all the advances in understanding the roles and the regulation of autophagy in health and disease realized during the past decade, the key question about the origin of the initial autophagosomal membranes remains largely unknown. Among the 16 autophagy-related (Atg) proteins composing the conserved machinery required for autophagy, Atg9 is the only integral membrane component and it is one of the first Atg proteins to be recruited to the phagophore assembly site (PAS) emphasizing its relevance in the early stages of autophagosome biogenesis. Because it is: intrinsically associated with lipid bilayers, Atg9 has all the prerequisites to be a major factor in regulating the supply of at least part of the membranes necessary for the formation and expansion of nascent autophagosomes.A reservoir of Atg9-containing vesicles and tubules provides the initial membranes necessary for autophagophore formation in yeast.

The mitochondrial contact site complex, a determinant of mitochondrial architecture

Mitochondria are organelles with a complex architecture. They are bounded by an envelope consisting of the outer membrane and the inner boundary membrane (IBM). Narrow crista junctions (CJs) link the IBM to the cristae. OMs and IBMs are firmly connected by contact sites (CS). The molecular nature of the CS remained unknown. Using quantitative high‐resolution mass spectrometry we identified a novel complex, the mitochondrial contact site (MICOS) complex, formed by a set of mitochondrial membrane proteins that is essential for the formation of CS. MICOS is preferentially located at the CJs. Upon loss of one of the MICOS subunits, CJs disappear completely or are impaired, showing that CJs require the presence of CS to form a superstructure that links the IBM to the cristae. Loss of MICOS subunits results in loss of respiratory competence and altered inheritance of mitochondrial DNA.

Regulation of endoplasmic reticulum turnover by selective autophagy

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy (‘ER-phagy’). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

Autophagic processes in yeast: mechanism, machinery and regulation.

Autophagy refers to a group of processes that involve degradation of cytoplasmic components including cytosol, macromolecular complexes, and organelles, within the vacuole or the lysosome of higher eukaryotes. The various types of autophagy have attracted increasing attention for at least two reasons. First, autophagy provides a compelling example of dynamic rearrangements of subcellular membranes involving issues of protein trafficking and organelle identity, and thus it is fascinating for researchers interested in questions pertinent to basic cell biology. Second, autophagy plays a central role in normal development and cell homeostasis, and, as a result, autophagic dysfunctions are associated with a range of illnesses including cancer, diabetes, myopathies, some types of neurodegeneration, and liver and heart diseases. That said, this review focuses on autophagy in yeast. Many aspects of autophagy are conserved from yeast to human; in particular, this applies to the gene products mediating these pathways as well as some of the signaling cascades regulating it, so that the information we relate is relevant to higher eukaryotes. Indeed, as with many cellular pathways, the initial molecular insights were made possible due to genetic studies in Saccharomyces cerevisiae and other fungi.

Atg9 cycles between mitochondria and the pre-autophagosomal structure in yeasts

Autophagy is a degradative process conserved among eukaryotic cells. It allows the elimination of cytoplasm including aberrant protein aggregates and damaged organelles. Accordingly, it is implicated in normal developmental processes and also serves a protective role in tumor suppression and elimination of invading pathogens, whereas defects in autophagy are associated with various human diseases including cancer and neurodegeneration. Atg proteins mediate the sequestration event that occurs at the preautophagosomal structure (PAS) by catalyzing the formation of double-membrane vesicles, termed autophagosomes. In Saccharomyces cerevisiae, the integral membrane protein Atg9 that is required for autophagy cycles through the PAS. Here, we demonstrate that Atg9 shuttles between this location and mitochondria. These data support a new model where mitochondria may provide at least part of the autophagosomal lipids and suggest a novel cellular function for this well-studied organelle.