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

Approaching the Molecular Mechanism of Autophagy

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

Reconstituting the Cvt pathway: An approach to unraveling autophagy [abstract]

Faculty Mentor: Dr. Silvia Jurisson, RadiochemistryAbstract only availableAutophagy, literally self-cannibalism, is a highly regulated catabolic process that is important in the maintenance of all eukaryotic cells. In humans, both excess and insufficient autophagy is linked to many diseases including cancer, Huntington's and Parkinson's diseases. Autophagy is the process in which defective and unwanted organelles along with cytoplasm are taken into an autophagosome and transported to the vacuole or lysosome to be degraded and used to build new macromolecules. Although most of the proteins involved in the autophagic pathway are known, their specific functions remain unknown. In order to better understand the molecular mechanisms of autophagy, we are using a specialized autophagic pathway found only in the yeast Saccharomyces cerevisiae: the Cytoplasm to vacuole targeting (Cvt) pathway. The Cvt pathway is used to deliver the inactive precursor of aminopeptidase1 (prApe1), to the vacuole. The pathway begins in the cytosol when prApe1 aggregates into dodecamers, which then form the Ape1 complex. This is followed by Atg19 binding to the Ape1 complex. Atg19 interacts with Atg11 (and possibly Atg8), which recruits autophagic membrane. These proteins along with a few others complete the Cvt vesicle, which then fuses with vacuolar membrane allowing Ape1 to be released into the lumen of the vacuole. We are attempting two different approaches to understand the Cvt pathway. In our first approach we are trying to reconstitute the Cvt pathway in Pichia pastoris, a related yeast strain in which the pathway does not occur. So far we have been successful in expressing S. cerevisiae's ape1 and atg19 genes in P. pastoris. In these strains the Ape1 complex aggregates and Atg19 co-localizes. We are still working on expressing atg11 in P. pastoris. Our second approach is to form the Cvt vesicle in vitro. In S. cerevisiae stable Ape1 complexes can be observed microscopically. Ape1 is also stable when co-localized with Atg19 as seen in the microscope. We are currently trying to purify the Ape1 complex, which is essential for using as a scaffold for in vitro formation of the Cvt vesicle

Reconstituting the Cvt pathway in pichia pastoris [abstract]

Abstract only availableIn yeast, autophagy is primarily a response to nutritional stress in which the cell sacrifices some of its cytoplasm and organelles in order to survive. Saccharomyces cerevisiae contains a specialized pathway for the delivery of certain proteins to the vacuole by selective autophagy called the Cytoplasm to vacuole targeting (Cvt) pathway. While autophagy is generally believed to be non-selective, the Cvt pathway is highly specific, transporting only a few selected proteins to the vacuole. The two known cargo proteins of the Cvt pathway are -mannosidase1 (Ams1) and the inactive precursor of aminopeptidase1 (prApe1). The pathway begins in the cytosol when prApe1 oligomerizes into dodecamers, which then aggregate to form the Ape1 complex. We are using the Ape1 aggregate as a model system for studying how protein aggregates are delivered to the vacuole/lysosome by constitutive and selective autophagy. We have introduced Ape1 from S. cerevisiae into the yeast Pichia pastoris in an attempt to reconstitute the Cvt pathway in this yeast. P. pastoris is related to S. cerevisiae, but does not contain the Cvt pathway. We are currently studying the requirements for the uptake of Ape1 into the vacuole in P. pastoris.McNair Scholars Progra

Creating a temperature sensitive mutant of Atg21 using intein [abstract]

Abstract only availableAutophagy is the highly regulated process by which a cell sequesters pieces of itself into vesicles called autophagosomes to recycle old components or to respond to nutritional stress. In addition, S. cerevisiae uses a specialize form of autophagy called the Cvt pathway to deliver the protease Aminopeptidase I (Ape1) to the vacuole. The Cvt pathway utilizes the same proteins and molecular mechanisms as autophagy. Atg 21 belongs to a novel family of phosphoinositide binding proteins and is essential for the Cvt pathway but not for nitrogen starvation-induced autophagy in S. cerevisiae. Based on previous studies combined with the location of Atg21 it has been proposed that the protein might be involved in generating new autophagic membrane or recruiting existing membrane to the site of sequestering vesicle formation but the exact function has yet to be determined. To further research the function of Atg21 a temperature sensitive inducible expression system was designed using Intein. Intein is a protein segment that can excise itself from a protein and rejoin the remaining portion (Extein) of the protein with a peptide bond. Intein mediated protein splicing occurs after mRNA translation. The presence of one of three conserved residues at the N-terminus (Ser, Thr, or Cys) and one of two dipeptides at the C-terminus (His-Asn or His-Gln) are essential for the splice junctions. Six cysteine residues (two near the N-terminus, two in the conserved domain, and two near the C-terminus) in Atg21 were selected as insertion sites for Intein. A temperature sensitive intein (TS-1/TS-19) was inserted at each different site on Atg21. Wild Type Intein was inserted in each site as a control for TS-1/TS-19 containing Atg21 cells. The expression of these Intein containing Atg21 cells has been verified by Western blotting and further experiments will now be conducted to verify the function of Atg21.Life Sciences Undergraduate Research Opportunity Progra