RESUMO
Macroautophagy, hereafter autophagy, is a major degradation pathway in eukaryotic systems that allows the removal of large intracellular structures such as entire organelles or protein aggregates, thus contributing to the homeostasis of cells and tissues. Autophagy entails the de novo formation of an organelle termed autophagosome, where a cup-shaped structure called isolation membrane nucleates in proximity of a cytoplasmic cargo material. Upon elongation and closure of isolation membranes, the mature autophagosome delivers the sequestered cargo into the lysosomal system for degradation. Among the factors for autophagosome formation are the autophagy-related (Atg) proteins belonging to the Atg8 conjugation system. In this system, the ubiquitin-like Atg8 protein is conjugated to the membrane lipid phosphatidylethanolamine present in autophagosomal membranes. Atg8 can also be removed from membranes by Atg4-mediated deconjugation. Here, we describe in vitro systems that recapitulate the enzymatic reactions occurring in vivo by presenting expression and purification strategies for all the components of the Saccharomyces cerevisiae Atg8 conjugation system. We also present protocols for in vitro Atg8 conjugation and deconjugation reactions employing small and giant unilamellar vesicles.
Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Lipossomas Unilamelares/química , Autofagia , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/isolamento & purificação , Proteína 7 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/isolamento & purificação , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos , Fosfatidiletanolaminas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/isolamento & purificação , Enzimas de Conjugação de Ubiquitina/metabolismo , Lipossomas Unilamelares/metabolismo , Proteína Vermelha FluorescenteRESUMO
The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), and its associated pathogen "Candidatus Liberibacter solanacearum" (Ca. L. solanacearum), the putative causal agent of zebra chip (ZC) disease in potatoes (Solanum tuberosum L.), were sampled in commercial potato fields and untreated control plots for 3 yr in multiple locations in Texas, Kansas, Nebraska, and Colorado. Populations of the potato psyllid varied across years and across potato growing regions. However, the percentage of potato psyllids infected with Ca. L. solanacearum although variable across years, was consistently highest in the Lower Rio Grande Valley of Texas (LRGV), the reported overwintering location for this pest. The numbers of Ca. L. solanacearum-infected psyllids collected on field traps and large nymphs counted on leaf samples were both positively correlated with the final percentage of ZC in tubers. In the LRGV, where vector and disease pressure is the highest, population levels of immature life stages of the psyllid and percentage of ZC differed greatly between commercial and untreated fields. These results show that the pest management program that was used can be effective at controlling development of the psyllid and ultimately reducing the incidence of ZC.
