Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4.

Coronaviruses replicate their genomes in association with rearranged cellular membranes. The coronavirus nonstructural integral membrane proteins (nsps) 3, 4 and 6, are key players in the formation of the rearranged membranes. Previously, we demonstrated that nsp3 and nsp4 interact and that their co-expression results in the relocalization of these proteins from the endoplasmic reticulum (ER) into discrete perinuclear foci.

An autophagy-independent role for LC3 in equine arteritis virus replication.

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus. Genome replication of EAV has been associated with modified intracellular membranes that are shaped into double-membrane vesicles (DMVs). We showed by immuno-electron microscopy that the DMVs induced in EAV-infected cells contain double-strand (ds)RNA molecules, presumed RNA replication intermediates, and are decorated with the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3).

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding.

Visualizing coronavirus RNA synthesis in time by using click chemistry.

Coronaviruses induce in infected cells the formation of replicative structures, consisting of double-membrane vesicles (DMVs) and convoluted membranes, where viral RNA synthesis supposedly takes place and to which the nonstructural proteins (nsp's) localize. Double-stranded RNA (dsRNA), the presumed intermediate in RNA synthesis, is localized to the DMV interior. However, as pores connecting the DMV interior with the cytoplasm have not been detected, it is unclear whether RNA synthesis occurs at these same sites.

Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication.

Coronaviruses (CoV), including SARS and mouse hepatitis virus (MHV), are enveloped RNA viruses that induce formation of double-membrane vesicles (DMVs) and target their replication and transcription complexes (RTCs) on the DMV-limiting membranes. The DMV biogenesis has been connected with the early secretory pathway. CoV-induced DMVs, however, lack conventional endoplasmic reticulum (ER) or Golgi protein markers, leaving their membrane origins in question.

Multiple roles of the cytoskeleton in autophagy.

Autophagy is involved in a wide range of physiological processes including cellular remodeling during development, immuno-protection against heterologous invaders and elimination of aberrant or obsolete cellular structures. This conserved degradation pathway also plays a key role in maintaining intracellular nutritional homeostasis and during starvation, for example, it is involved in the recycling of unnecessary cellular components to compensate for the limitation of nutrients. Autophagy is characterized by specific membrane rearrangements that culminate with the formation of large cytosolic double-membrane vesicles called autophagosomes.

Harpooning the Cvt complex to the phagophore assembly site.

Autophagy is a catabolic process employed by eukaryotes to degrade and recycle intracellular components. When this pathway is induced by starvation conditions, part of the cytoplasm and organelles are sequestered into double-membrane vesicles called autophagosomes, and delivered into the lysosome/vacuole for degradation. In addition to the random bulk elimination of cytoplasmic contents, the selective removal of specific cargo molecules has also been described.

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.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.

Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast.

Autophagy is a conserved degradative pathway that is induced in response to various stress and developmental conditions in eukaryotic cells. It allows the elimination of cytosolic proteins and organelles in the lysosome/vacuole. In the yeast Saccharomyces cerevisiae, the integral membrane protein Atg9 (autophagy-related protein 9) cycles between mitochondria and the preautophagosomal structure (PAS), the nucleating site for formation of the sequestering vesicle, suggesting a role in supplying membrane for vesicle formation and/or expansion during autophagy.

Autophagy in organelle homeostasis: peroxisome turnover.

When cells are confronted with an insufficient supply of nutrients in their extracellular fluid, they may begin to cannibalize some of their internal proteins as well as whole organelles for reuse in the synthesis of new components. This process is termed autophagy and it involves the formation of a double-membrane structure within the cell, which encloses the material to be degraded into a vesicle called an autophagosome. The autophagosome subsequently fuses with a lysosome/vacuole whose hydrolytic enzymes degrade the sequestered organelle.

Atg11 directs autophagosome cargoes to the PAS along actin cables.

For more than 40 years, autophagy has been almost exclusively studied as a cellular response that allows adaptation to starvation situations. In nutrient-deprived conditions, cytoplasmic components and organelles are randomly sequestered into double-membrane vesicles called autophagosomes, creating the notion that this pathway is a nonselective process (reviewed in Refs 1, 2). Recent results, however, have demonstrated that under certain circumstances, cargoes such as protein complexes, organelles and bacteria can be selectively and exclusively incorporated into double-membrane vesicles.

The Hansenula polymorpha ATG25 gene encodes a novel coiled-coil protein that is required for macropexophagy.

We have isolated the Hansenula polymorpha ATG25 gene, which is required for glucose-induced selective peroxisome degradation by macropexophagy. ATG25 represents a novel gene that encodes a 45 kDa coiled-coil protein. We show that this protein colocalizes with Atg11 on a small structure, which most likely represents the pre-autophagosomal structure (PAS).

Hansenula polymorpha Vam7p is required for macropexophagy.

We have analyzed the functions of two vacuolar t-SNAREs, Vam3p and Vam7p, in peroxisome degradation in the methylotrophic yeast Hansenula polymorpha. A Hp-vam7 mutant was strongly affected in peroxisome degradation by selective macropexophagy as well as non-selective microautophagy. Deletion of Hp-Vam3p function had only a minor effect on peroxisome degradation processes.

The actin cytoskeleton is required for selective types of autophagy, but not nonspecific autophagy, in the yeast Saccharomyces cerevisiae.

Autophagy is a catabolic multitask transport route that takes place in all eukaryotic cells. During starvation, cytoplasmic components are randomly sequestered into huge double-membrane vesicles called autophagosomes and delivered into the lysosome/vacuole where they are destroyed. Cells are able to modulate autophagy in response to their needs, and under certain circumstances, cargoes such as aberrant protein aggregates, organelles and bacteria can be selectively and exclusively incorporated into autophagosomes.

Atg8 is essential for macropexophagy in Hansenula polymorpha.

We have isolated a peroxisome-degradation-deficient (pdd) mutant of the methylotrophic yeast Hansenula polymorpha via gene tagging mutagenesis. Sequencing revealed that the mutant was affected in the HpATG8 gene. HpAtg8 is a protein with high sequence similarity to both Pichia pastoris and Saccharomyces cerevisiae Atg8 and appeared to be essential for selective peroxisome degradation (macropexophagy) and nitrogen-limitation induced microautophagy.

Old yellow enzyme confers resistance of Hansenula polymorpha towards allyl alcohol.

In the methylotrophic yeast, Hansenula polymorpha, peroxisomes are formed during growth on methanol as sole carbon and energy source and contain the key enzymes for its metabolism, one of the major enzymes being alcohol oxidase (AO). Upon a shift of these cells to glucose-containing medium, peroxisomes become redundant for growth and are rapidly degraded via a highly selective process designated macropexophagy. H.