Chapter title |
Identifying a rab effector on the macroautophagy pathway.
|
---|---|
Chapter number | 10 |
Book title |
Rab GTPases
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2569-8_10 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2568-1, 978-1-4939-2569-8
|
Authors |
Juan Wang, Serena Cervantes, Saralin Davis, Susan Ferro-Novick, Wang, Juan, Cervantes, Serena, Davis, Saralin, Ferro-Novick, Susan |
Abstract |
Rab GTPases are key regulators of membrane traffic. The Rab GTPase Ypt1 is essential for endoplasmic reticulum (ER)-Golgi traffic, intra-Golgi traffic, and the macroautophagy pathway. To identify effectors on the macroautophagy pathway, known autophagy-related genes (Atg genes) required for macroautophagy were tagged with GFP and screened for mislocalization in the ypt1-2 mutant. At the pre-autophagosomal structure (PAS), the localization of the serine/threonine kinase Atg1 was affected in the ypt1-2 mutant. We then used an in vitro binding assay to determine if Atg1 and Ypt1 physically interact with each other and co-immunoprecipitation experiments were performed to address if Atg1 preferentially interacts with the GTP-bound form of Ypt1. |
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