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Rab GTPases pp 117-125 | Cite as

Identifying a Rab Effector on the Macroautophagy Pathway

  • Juan Wang
  • Serena Cervantes
  • Saralin Davis
  • Susan Ferro-NovickEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)

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.

Key words

Ypt1 Rab GTPase Effector Atg1 Macroautophagy 

Notes

Acknowledgements

Salary support for J.W., S.C., S.D., and S.F.-N. was provided by the Howard Hughes Medical Institute.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Juan Wang
    • 1
  • Serena Cervantes
    • 1
  • Saralin Davis
    • 1
  • Susan Ferro-Novick
    • 1
    Email author
  1. 1.Department of Cellular and Molecular MedicineHoward Hughes Medical Institute, University of California at San DiegoLa JollaUSA

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