Making the Cvt pathway/autophagy in vitro

Abstract only availableAutophagy, Greek for “self eating”, occurs in all eukaryotic cells to remove damaged or unwanted organelles or to provide a source of nutrients during starvation. In autophagy, a double membrane surrounds a cluster of contents, damaged organelles, bulk cytoplasm, and aminopeptidase I (Ape1), forming an autophagic vesicle to be sent to the vacuole. A unique Cvt pathway occurring in Saccharomyces cerevisiae, includes a membrane encapsulation of only Ape1 to be transported directly to the vacuole. Most of the proteins needed for autophagy and the Cvt pathway have been identified, but their roles have yet to be determined. In the Cvt pathway, the Ape1 aggregates to form a dodecamer before forming a Cvt complex by combining with the protein Atg19. The Cvt complex affixes to the autophagic membrane presumably with the aid of Atg11 and Atg8. An array of proteins, Atg9-Atg2 complex, Atg1, and Pi3-kinase complex, help complete the formation of the autophagic vesicle encompassing Ape1. The autophagic vesicle then fuses with the vacuole releasing the Ape1 into the lumen of the vacuole. Until now, only whole cells have been used to examine autophagy. Due to the complexity of the whole cell, the functions of the all proteins needed for autophagy have not been determined. Our main goal is to be able to construct the Cvt pathway in vitro. We have begun inserting the APE1 gene from S. cerevisiae into another strain of yeast, Pichia pastoris, where the Cvt pathway does not occur. Once the proteins are expressed in P. pastoris, we will study the interaction of Ape1 with other Cvt proteins. From P. pastori we will extract Cvt proteins for examining in a test tube. While in vitro, it will be possible to determine the molecular function each protein contributes to autophagy. A better understanding of the process of autophagy will be beneficial to understanding and treatment of many diseases such as cancer, liver disease, muscular disorder, neurodegeneration and bacteria infections.Life Sciences Undergraduate Research Opportunity Progra

Yeast homotypic vacuole fusion requires the Ccz1–Mon1 complex during the tethering/docking stage

The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1–Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1–Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1–Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1–Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion

Autophagy: A Forty-Year Search for a Missing Membrane Source

Autophagy is central to diverse biological processes in eukaryotes including animal development and cellular survival, and also to neurodegenerative diseases, but the origin of the membranes that make up autophagic vesicles is unknown

Approaching the Molecular Mechanism of Autophagy

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73484/1/j.1600-0854.2001.20802.x.pd

Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells

Apoptotic and autophagic cell death have been implicated, on the basis of morphological and biochemical criteria, in neuronal loss occurring in neurodegenerative diseases and it has been shown that they may overlap. We have studied the relationship between apoptosis and autophagic cell death in cerebellar granule cells (CGCs) undergoing apoptosis following serum and potassium deprivation. We found that apoptosis is accompanied by an early and marked proliferation of autophagosomal-lysosomal compartments as detected by electron microscopy and immunofluorescence analysis. Autophagy is blocked by hrIGF-1 and forskolin, two well-known inhibitors of CGC apoptosis, as well as by adenovirus-mediated overexpression of Bcl-2. 3-Methyladenine (3-MA) an inhibitor of autophagy, not only arrests this event but it also blocks apoptosis. The neuroprotective effect of 3-MA is accompanied by block of cytochrome c (cyt c) release in the cytosol and by inhibition of caspase-3 activation which, in turn, appears to be mediated by cathepsin B, as CA074-Me, a selective inhibitor of this enzyme, fully blocks the processing of pro-caspase-3. Immunofluorescence analysis demonstratesd that cathepsin B, normally confined inside the lysosomal-endosomal compartment, is released during apoptosis into the cytosol where this enzyme may act as an execution protease. Collectively, these observations indicate that autophagy precedes and is causally connected with the subsequent onset of programmed death

Subversion of Cellular Autophagosomal Machinery by RNA Viruses

Infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral RNA replication and whose origin is unknown. Here, we show that several hallmarks of cellular autophagosomes can be identified in poliovirus-induced vesicles, including colocalization of LAMP1 and LC3, the human homolog of Saccharomyces cerevisiae Atg8p, and staining with the fluorophore monodansylcadaverine followed by fixation. Colocalization of LC3 and LAMP1 was observed early in the poliovirus replicative cycle, in cells infected with rhinoviruses 2 and 14, and in cells that express poliovirus proteins 2BC and 3A, known to be sufficient to induce double-membraned vesicles. Stimulation of autophagy increased poliovirus yield, and inhibition of the autophagosomal pathway by 3-methyladenine or by RNA interference against mRNAs that encode two different proteins known to be required for autophagy decreased poliovirus yield. We propose that, for poliovirus and rhinovirus, components of the cellular machinery of autophagosome formation are subverted to promote viral replication. Although autophagy can serve in the innate immune response to microorganisms, our findings are inconsistent with a role for the induced autophagosome-like structures in clearance of poliovirus. Instead, we argue that these double-membraned structures provide membranous supports for viral RNA replication complexes, possibly enabling the nonlytic release of cytoplasmic contents, including progeny virions, from infected cells

The Radish Gene Reveals a Memory Component with Variable Temporal Properties

Memory phases, dependent on different neural and molecular mechanisms, strongly influence memory performance. Our understanding, however, of how memory phases interact is far from complete. In Drosophila, aversive olfactory learning is thought to progress from short-term through long-term memory phases. Another memory phase termed anesthesia resistant memory, dependent on the radish gene, influences memory hours after aversive olfactory learning. How does the radish-dependent phase influence memory performance in different tasks? It is found that the radish memory component does not scale with the stability of several memory traces, indicating a specific recruitment of this component to influence different memories, even within minutes of learning