A lifespan-extending form of autophagy employs the vacuole-vacuole fusion machinery

While autophagy is believed to be beneficial for lifespan extension, it is controversial which forms or aspects of autophagy are the responsible ones. We addressed this by analyzing the lifespan of yeast autophagy mutants under caloric restriction, a longevity manipulation. Surprisingly, we discovered that the majority of proteins involved in macro-autophagy and several forms of micro-autophagy were dispensable for lifespan extension. The only autophagy protein that is critical for lifespan extension was Atg15p, a lipase that is located in the endoplasmic reticulum (ER) and transported to vacuoles for disintegrating membranes of autophagic bodies. We further found that vacuole-vacuole fusion was required for lifespan extension, which was indicated by the shortened lifespan of mutants missing proteins (ypt7Δ, nyv1Δ, vac8Δ) or lipids (erg6Δ) involved in fusion. Since a known function of vacuole-vacuole fusion is the maintenance of the vacuole membrane integrity, we analyzed aged vacuoles and discovered that aged cells had altered vacuolar morphology and accumulated autophagic bodies, suggesting that certain forms of autophagy do contribute to longevity. Like aged cells, erg6Δ accumulated autophagic bodies, which is likely caused by a defect in lipase instead of proteases due to the existence of multiple vacuolar proteases. Since macro-autophagy is not blocked by erg6Δ, we propose that a new form of autophagy transports Atg15p via the fusion of vacuoles with vesicles derived from ER, and we designate this putative form of autophagy as secretophagy. Pending future biochemical studies, the concept of secretophagy may provide a mechanism for autophagy in lifespan extension.     

A current view of molecular dissection in autophagy machinery

Journal of Physiology and Biochemistry - Macroautophagy (hereafter called autophagy) is a highly conserved lysosomal pathway for catabolism of intracellular material in eukaryotic cells. Autophagy...     

Are nidoviruses hijacking the autophagy machinery?

Autophagy is an intracellular catabolic transport route conserved among all eukaryotic cells. It has multiple important physiological functions, one of which is to act as an immune mechanism against intracellular microbes.1,2 Bacteria and viruses targeted for destruction are sequestered into large double-membrane vesicles called autophagosomes and subsequently delivered to the lysosomes where they are consumed by resident hydrolases. Unfortunately, conserved cellular pathways are often exploited by pathogens to facilitate their entry or replication, and autophagy is no exception. It has become clear that certain bacteria and viruses subvert this process to their advantage.3 Nidoviruses, which comprise coronaviruses and arteriviruses, might be among the successful. Long recognized as the cause of veterinary infections, this group of enveloped, positive-stranded RNA viruses is currently of considerable interest due to the emergence of new human coronaviruses, especially the severe acute respiratory syndrome (SARS)-coronavirus. In this mini-review, we will summarize the so far limited number of studies that have demonstrated that double-membrane vesicles resembling autophagosomes are the sites of nidoviruses replication. In addition, we will discuss how the formation of these large vesicles might be induced.     

Viruses and the autophagy machinery

Autophagy is a major intracellular pathway for degradation and recycling of long-lived proteins and cytoplasmic organelles that plays an essential role in maintenance of homeostasis in response to starvation and other cellular stresses. Autophagy is also important for a variety of other processes including restriction of intracellular pathogen replication. Our understanding of the fascinating relationship between viruses and the autophagy machinery is still in its infancy but it is clear that autophagy is a newly recognized facet of innate and adaptive immunity against viral infection. Although the autophagy pathway is emerging as a component of host defense, certain viruses have developed strategies to counteract these antiviral mechanisms, and others appear to have co-opted the autophagy machinery as proviral host factors favoring viral replication. The complex interplay between autophagy and viral infection will be discussed in this review.     

The machinery of vesicle fusion

The compartmentalization of eukaryotic cells is reliant on the fidelity of vesicle-mediated intracellular transport. Vesicles deliver their cargo via membrane fusion, a process requiring membrane tethers, Sec1/Munc18 (SM) proteins, and SNAREs. These components function in concert to ensure that membrane fusion is efficient and accurate, but the mechanisms underlying their cooperative action are still in many respects mysterious. In this brief review, we highlight recent progress toward a more integrative understanding of the vesicle fusion machinery. We focus particular attention on cryo-electron microscopy structures of intact multisubunit tethers in complex with SNAREs or SM proteins, as well as a structure of an SM protein bound to multiple SNAREs. The insights gained from this work emphasize the advantages of studying the fusion machinery intact and in context.     

Palmitoylation of the synaptic vesicle fusion machinery

The fusion of synaptic vesicles with the pre-synaptic plasma membrane mediates the secretion of neurotransmitters at nerve terminals. This pathway is regulated by an array of protein–protein interactions. Of central importance are the soluble NSF ( N -ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP25, which are associated with the pre-synaptic plasma membrane and vesicle-associated membrane protein (VAMP2), a synaptic vesicle SNARE. Syntaxin 1, SNAP25 and VAMP2 interact to form a tight complex bridging the vesicle and plasma membranes, which has been suggested to represent the minimal membrane fusion machinery. Synaptic vesicle fusion is stimulated by a rise in intraterminal Ca²⁺ levels, and a major Ca²⁺ sensor for vesicle fusion is synaptotagmin I. Synaptotagmin is likely to couple Ca²⁺ entry to vesicle fusion via Ca²⁺-dependent and independent interactions with membrane phospholipids and the SNARE proteins. Intriguingly, syntaxin 1, SNAP25, VAMP2 and synaptotagmin I have all been reported to be modified by palmitoylation in neurons. In this review, we discuss the mechanisms and dynamics of palmitoylation of these proteins and speculate on how palmitoylation might contribute to the regulation of synaptic vesicle fusion.     

Topological arrangement of the intracellular membrane fusion machinery

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) form a four-helix coiled-coil bundle that juxtaposes two bilayers and drives a basal level of membrane fusion. The Sec1/Munc18 (SM) protein binds to its cognate SNARE bundle and accelerates the basal fusion reaction. The question of how the topological arrangement of the SNARE helices affects the reactivity of the fusion proteins remains unanswered. Here we address the problem for the first time in a reconstituted system containing both SNAREs and SM proteins. We find that to be fusogenic a SNARE topology must support both basal fusion and SM stimulation. Certain topological combinations of exocytic SNAREs result in basal fusion but cannot support SM stimulation, whereas other topologies support SM stimulation without inducing basal fusion. It is striking that of all the possible topological combinations of exocytic SNARE helices, only one induces efficient fusion. Our results suggest that the intracellular membrane fusion complex is designed to fuse bilayers according to one genetically programmed topology.     

Interplay between the cellular autophagy machinery and positive-stranded RNA viruses

Autophagy is a conserved cellular process that acts as a key regulator in maintaining cellular homeostasis. Recent studies implicate an important role for autophagy in infection and immunity by removing invading pathogens and through modulating innate and adaptive immune responses. However, several pathogens, notably some positive-stranded RNA viruses, have subverted autophagy to their own ends. In this review, we summarize the current understanding of how viruses with a positive-stranded RNA genome interact with the host autophagy machinery to control their replication and spread. We review the mechanisms underlying the induction of autophagy and discuss the pro- and anti-viral functions of autophagy and the potential mechanisms involved.     

Granuphilin molecularly docks insulin granules to the fusion machinery

The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin-null mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin-null beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.     

Interplay between lipids and the proteinaceous membrane fusion machinery

For membrane fusion to occur, opposed lipid bilayers initially establish a fusion pore, often followed by complete mixing of the fusing membranes. Contemporary views suggest that during fusion lipid bilayers are continuous passive platforms that are disrupted and remodeled by catalytic proteins. Some models propose that even the architecture and composition of the fusion pore might be dominated by proteins rather than lipids. Hence, lipids have no regulatory contribution to this process; they simply adapt their shape passively for filling space between otherwise autonomous protein machineries.However, an increasing number of experimental findings indicate that membrane fusion critically depends on a variety of lipids and lipid derivatives. Therefore, a purely proteocentric view describes fusion mechanisms insufficiently. Instead, lipids have functions probably at different levels, as (i) a general influence on the propensity of lipid bilayers to fuse, (ii) a role in recruiting exocytotic proteins to the plasma membrane, (iii) a role in organizing membrane domains for fusion and (iv) direct regulatory effects on fusion protein complexes. In this review we have made an attempt to bring together the large body of evidence supporting a major role for lipids in membrane fusion either directly or indirectly.     

Species‐specific impact of the autophagy machinery on Chikungunya virus infection

Chikungunya virus (CHIKV) is a recently re‐emerged arbovirus that triggers autophagy. Here, we show that CHIKV interacts with components of the autophagy machinery during its replication cycle, inducing a cytoprotective effect. The autophagy receptor p62 protects cells from death by binding ubiquitinated capsid and targeting it to autophagolysosomes. By contrast, the human autophagy receptor NDP52—but not its mouse orthologue—interacts with the non‐structural protein nsP2, thereby promoting viral replication. These results highlight the distinct roles of p62 and NDP52 in viral infection, and identify NDP52 as a cellular factor that accounts for CHIKV species specificity.     

The protein machinery of vesicle budding and fusion.

A general protein machinery that buds and fuses transport vesicles is harnessed to generate the complex web of intracellular transport pathways critical for such diverse processes as cell growth, endocytosis, hormone release, and neurotransmission. With this appreciation, the challenge of understanding the precise molecular mechanisms of these many facets of cell biology has been reduced to a series of problems in protein structure and chemistry.