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Implications of a genomic search for autophagy-related genes in trypanosomatids D.J. Rigden*1 , M. Herman†, S. Gillies* and P.A.M. Michels† *School of Biological Sciences, University of Liverpool, Liverpool, U.K., and †Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Universite´ catholique de Louvain, Brussels, Belgium
Abstract Autophagy is the process by which cellular components are directed to and degraded in the vacuole or lysosome and has been studied largely in yeasts. We present here an in silico genomic analysis of trypanosomatid autophagy aimed at highlighting similarities and differences with autophagy in other organisms. Less than half of the yeast autophagy-related proteins examined have certain putative orthologues in trypanosomatids. A cytosol-to-vacuole transport system is clearly lacking in these organisms. Other absences are even more unexpected and have implications for our understanding of the molecular mechanisms of autophagy. The results are consistent with taxon-specific addition of components to a core autophagy machinery during evolution.
Introduction Autophagy is the targeting of cellular components to the vacuole or lysosome where they are degraded [1,2]. Such components include bulk cytoplasm, aberrant protein aggregates or entire cellular organelles that are damaged or surplus to requirements. Recently, invading pathogenic bacteria have been added to the list of cargoes disposed off in this way [3] and viruses may also be subject to autophagic processing. The list of physiological roles of autophagy grows ever longer and already contains contributions to normal cellular development and differentiation, lifespan extension and possible programmed cell death. Human diseases linked to autophagy include cancer, cardiomyopathy and various neurodegenerative disorders [4]. Autophagy may be divided into micro-autophagy, in which the cytoplasm is directly sequestered by the lysosomal/vacuolar membrane, and the much better understood macro-autophagy (often called simply autophagy). During macro-autophagy, vesicles called autophagosomes are formed from membranes whose origin remains unknown. These vesicles sequester the component in question and effect delivery by fusion with the vacuole or lysosome. Autophagy is related to, and shares several proteins with the CVT (cytosol-to-vacuole transport) pathway by which a minority of vacuole-resident hydrolases are delivered to that organelle [5]. Historically, most of our understanding of autophagy has come from studies of yeasts and, in particular, from genetic screens for mutants defective in autophagy or related processes. For example, methylotrophic yeasts, such as Hansenula polymorpha and Pichia pastoris, rapidly change their peroxisome populations when responding to changing Key words: autophagy, cytosol-to-vacuole transport (CVT), database searching, genome census, pexophagy, trypanosomatid. Abbreviations used: ATG, autophagy-related; CVT, cytosol-to-vacuole transport; PRB1, protease B1. 1 To whom correspondence should be addressed (email
[email protected]).
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nutritional circumstances such as a switch in availability as carbon source from methanol to glucose (or vice versa). This is achieved by autophagy of the original peroxisomes, a process called pexophagy, and by synthesis of new peroxisomes with a different complement of enzymes [6]. Both micro- and macro-pexophagy occur in these yeasts and in the model organism Saccharomyces cerevisiae. Autophagy is well known in mammalian cells and has been demonstrated in plants. Homologues to some autophagyimplicated yeast genes, but not others, may be found in other eukaryotes and this has stimulated the study of individual components of the pathway in other organisms. However, the opportunity, offered by the increasing availability of complete genome sequences, to predict the overall structure of the autophagy pathway in these and other organisms seems not to have been taken. Such surveys would help delineate common, core features of autophagy and differentiate them from lineage-specific additions, thereby enabling a fuller understanding of the evolution of this important cellular phenomenon. We have carried out a genomic census of trypanosomatidal putative orthologues of autophagy-related yeast genes. Standard database searches were supplemented wherever necessary with more sensitive methods. Specific evidence for orthology, including clustering in phylogenetic trees [7], was also sought, particularly in cases where only a distant relationship between the yeast gene and the most similar trypanosomatidal sequence was evident. Our choice of the trypanosomatids was not made just in view of the availability of three (near-) complete genomes (Trypanosoma brucei, Trypanosoma cruzi and Leishmania major; see http://www. genedb.org), a factor significantly aiding the analysis, but also with a specific hypothesis in mind regarding autophagy in these organisms. All three of these pathogens have complex life cycles involving insect vectors and mammalian hosts. In common with other kinetoplastids, they possess an unusual
Mechanistic and Functional Studies of Proteins
organelle, the glycosome, a relative of the peroxisome, in which enzymes of several metabolic pathways are sequestered [8]. Most prominent of these are those catalysing a large part of glycolysis, upon which these organisms are largely dependent in their mammalian stages. Could it be that turnover of glycosomes, ‘glycophagy’, accompanies the adaptation from one set of environmental circumstances to another during parasites’ life cycles, just as yeasts adapt to different carbon sources?
Results and discussion Experimental results regarding autophagy and related processes in trypanosomatids are still limited. Electron microscopy data of different stages are suggestive of autophagy [8a]. Our own studies, for example the association of glycosomes with enlarged lysosomes during T. brucei differentiation from bloodstream to insect forms, are consistent with the existence of ‘glycophagy’ (M. Herman and P. Michels, unpublished work). Such limited data could usefully be complemented by a genomic analysis. Except for some proteins acting in general vesicle fusion, whose presence in trypanosomatids may safely be assumed, we sought putative orthologues of all autophagy-implicated yeast proteins, a list of 40 sequences. After the introduction of a unified nomenclature [9], most of these are referred to as ATG (autophagy-related) proteins. Many could be straightforwardly identified as the top hits by BLAST [10] database searching, where other homologues were absent or separated by tens of orders of magnitude in E value. Similarly, many yeast proteins lacked any evident homologue in the trypanosomatid databases, even after more sensitive profile-based searches [10]. Between these situations, in only two cases, ATG6 and ATG15, were homologues found, but evidence for orthology was inconclusive. In the case of ATG1, many homologous sequences were visible, but a putative orthologue could not be discerned even after phylogenomic analysis. The first, remarkable result of the census was that, of the 40 sought, at most 20 orthologues of yeast autophagy-implicated proteins exist in trypanosomatids. We are confident that this is not an issue of the sensitivity of our search, since we employed sensitive profile-based methods and many putative orthologues were very readily identified. In addition, the coordinated absence of orthologues to functionally related yeast proteins (see below) supports the accuracy of our results. Among the 20 found are the apparently most important participants in autophagy. For example, the protein kinase TOR (target of rapamycin), important for the induction of autophagy, is present in trypanosomatids, as are ATG8 and the proteins that modify its structure [11], as key players in the expansion and completion of the autophagosomes. We interpret the presence of these putative orthologues to imply that autophagy (and indeed ‘glycophagy’) is genomically viable in trypanosomatids. On the other hand, the absence of half the proteins sought could mean that the roles of the yeast proteins are not required for a functioning autophagy
pathway. Alternatively, gene displacement [12] may have occurred, in which an unrelated protein takes over the role of the original participant. Likely candidates in the latter category are the two vacuolar yeast proteases, PRB1 (protease B1) and PEP4 (vacuolar aspartyl protease, also known as proteinase A), that help to degrade the components delivered by autophagy, neither of which appears to be present in trypanosomatids. Since proteases are numerous in the genome, enzymes of a different class could easily have assumed the roles of PRB1 and PEP4 in trypanosomatids. Two patterns within the absent proteins offer both insight into the global structure of trypanosomatidal autophagy and support for the accuracy of our census. In addition to sharing components with autophagy, the CVT process in yeasts employs several CVT-specific proteins, acting at diverse steps of the pathway. These proteins ATG11, ATG19, ATG20, ATG21, ATG23 and ATG27 are all absent from trypanosomatids, strongly suggesting that CVT does not occur in these organisms and implying that the orthologue of the hydrolase APE1 (aminopeptidase I), delivered to the yeast vacuole by CVT, arrives at the trypanosomatidal lysosome by conventional means. Most striking of the trypanosomatidal absences are those of the components of one of the two ubiquitin-like systems acting during vesicle expansion and completion [11]. Using this system, activated ATG12 is conjugated to ATG5 by ATG10, with the resultant conjugate forming a large complex through interaction with ATG16. The absence of this system in trypanosomatids is remarkable since various lines of evidence support a key role of the ATG12–ATG5/ATG16 complex both in vesicle expansion and in imposing curvature upon the nascent autophagosomes [2]. Either this hypothesis is wrong, or a radically different mechanism is operating in trypanosomatids.
Conclusions Surprisingly, at most half of the yeast autophagy-implicated proteins have putative orthologues in trypanosomatids. While some of the absences correspond to components of the CVT pathway, hitherto only described in S. cerevisiae, others are more unexpected, with implications for our understanding of the molecular mechanisms of autophagy. While similar surveys of other eukaryotes are required to achieve a complete picture, our results are consistent with taxon-specific addition of components to a minimal core. Future experimental data will show whether further trypanosomatidal proteins, lacking yeast orthologues, act in autophagy and related phenomena.
The bioinformatics analysis was supported by a Research Development Fund award from the University of Liverpool. The glycophagy research in the Brussels laboratory is financially supported through grants from the Belgian ‘Fonds de la Recherche Scientifique Medicale’ ´ (FRSM) and Interuniversity Attraction Poles (IAP) – Federal C 2005
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Office for Scientific, Technical and Cultural Affairs. M.H. acknowledges a PhD scholarship from the ‘Fonds pour la formation a` la Recherche dans l’Industrie et dans l’Agriculture’ (FRIA).
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