Autophagy in Trypanosoma brucei: Amino Acid Requirement and Regulation during Different Growth Phases

Remo S Schmidt,Peter Bütikofer,Ger Van Zandbergen

doi:10.1371/journal.pone.0093875

Abstract

Autophagy in the protozoan parasite, Trypanosoma brucei, may be involved in differentiation between different life cycle forms and during growth in culture. We have generated multiple parasite cell lines stably expressing green fluorescent protein- or hemagglutinin-tagged forms of the autophagy marker proteins, TbAtg8.1 and TbAtg8.2, in T. brucei procyclic forms to establish a trypanosome system for quick and reliable determination of autophagy under different culture conditions using flow cytometry. We found that starvation-induced autophagy in T. brucei can be inhibited by addition of a single amino acid, histidine, to the incubation buffer. In addition, we show that autophagy is induced when parasites enter stationary growth phase in culture and that their capacity to undergo starvation-induced autophagy decreases with increasing cell density.

Highlights

When eukaryotic cells encounter stress conditions, such as nutrient starvation, they begin to recycle cellular contents using macroautophagy, referred to as autophagy
yellow fluorescent protein (YFP)- and Ty1-tagged forms of TbAtg8.2 constitutively expressed in T. brucei procyclic forms were described as suitable markers to monitor autophagy [27]
We show that N-terminally GFPtagged TbAtg8.1 is suited to monitor autophagy in T. brucei

Summary

Introduction

When eukaryotic cells encounter stress conditions, such as nutrient starvation, they begin to recycle cellular contents using macroautophagy, referred to as autophagy During this process, cells engulf cytoplasm containing proteins to be degraded by a double membrane structure, the autophagosome. Soon after synthesis [7], Atg is cleaved by the cysteine protease, Atg, to expose a glycine residue at the protein’s Cterminus, which upon induction of autophagy becomes covalently modified by phosphatidylethanolamine (PE), catalyzed by Atg and Atg3 [7,8] This modification changes the biophysical properties of Atg leading to altered sub-cellular distribution: the small, hydrophilic, 13.5 kDa protein receives a hydrophobic membrane anchor, causing re-localization from the cytosol to autophagosomes. The sub-population of Atg located on the outer membrane is released by Atg4 [7], whereas the Atg on the inner membrane is degraded by lysosomal hydrolases together with the inner membrane [10]

Methods

Results

Conclusion