المؤلفون: Usha Nair, Daniel J. Klionsky, Roswitha Krick, Michael Thumm

المصدر: Autophagy. 7:1546-1550

مصطلحات موضوعية: Autophagosome, Autophagy-Related Protein 8 Family, Saccharomyces cerevisiae Proteins, Recombinant Fusion Proteins, ATG8, medicine.medical_treatment, Green Fluorescent Proteins, Saccharomyces cerevisiae, Vacuole, Biology, Aminopeptidases, Phagosomes, Autophagy, Protocol, medicine, Molecular Biology, Phagosome, Protease, Cell Biology, Cell biology, Biochemistry, Biological Assay, Microtubule-Associated Proteins, Biogenesis

الوصف: Perhaps the most complex step of macroautophagy is the formation of the double-membrane autophagosome. The majority of the autophagy-related (Atg) proteins are thought to participate in nucleation and expansion of the phagophore, and/or the completion of this compartment. Monitoring this part of the process is difficult, and typically involves electron microscopy analysis; however, unless three-dimensional tomography is performed, even this method cannot be used to easily determine if the phagophore is completely enclosed. Accordingly, a complementary approach is to examine the accessibility of sequestered cargo to exogenously added protease. This type of protease protection analysis has been used to monitor the formation of cytoplasm-to-vacuole targeting (Cvt) vesicles and autophagosomes by examining the protease sensitivity of precursor aminopeptidase I (prApe1). For determining the status of autophagosomes formed during nonselective autophagy, however, prApe1 is not the best marker protein. Here, we describe an alternative method for examining autophagosome completion using GFP-Atg8 as a marker for protease protection.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::144976d94495b45bb49b4abd7ae12ca8Test
https://doi.org/10.4161/auto.7.12.18424Test

المؤلفون: Stephan vom Dahl, Tanja Prick, Michael Thumm, Dieter Häussinger

المصدر: Autophagy. 2:241-243

مصطلحات موضوعية: Protein Denaturation, Saccharomyces cerevisiae Proteins, Osmotic shock, Proteolysis, ATG8, Biology, Models, Biological, Microtubule, Gene Expression Regulation, Fungal, Autophagy, medicine, Animals, Humans, Molecular Biology, Sirolimus, Starvation, medicine.diagnostic_test, Mechanism (biology), Autophagy-Related Protein 8 Family, Cell Biology, Water-Electrolyte Balance, Yeast, Cell biology, Biochemistry, Mitogen-Activated Protein Kinases, medicine.symptom, Microtubule-Associated Proteins, Gene Deletion

الوصف: The mechanisms of regulation of autophagy are still obscure. In mammalian liver, starvation-induced autophagic proteolysis is regulated by the cellular hydration state in a microtubule- and p38MAPK-dependent way. Recent work shows that in yeast, loss of Hog1, the yeast orthologue of p38MAPK, leads to osmosensitivity of starvation-induced autophagy (Prick et al. Biochem J 2006; 394:153-61), pointing to an evolutionarily conserved mechanism. In this addendum further experiments from hog1delta yeast cells are shown, which support the hypothesis that starvation- and rapamycin-induced autophagy processes differ in their susceptibility to osmotic stress. The potential mechanisms are discussed. Addendum to:In Yeast, Loss of Hog1 Leads to Osmosensitivity of AutophagyT. Prick, M. Thumm, K. Kohrer, D. Haussinger and S. vom DahlBiochem J 2006; 394:153-61

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b2764ea5d8522a9d897c7ef23793aa6cTest
https://doi.org/10.4161/auto.2743Test