Comparative in-silico analysis of autophagy-related genes (ATG) in
ciliates
Erhan Aslan1,2, Nurçin Küçükoğlu 1,2, Muhittin Arslanyolu1,2
1
Graduate School of Science, Molecular Biology Program, Anadolu University, 26470, Eskişehir,
Turkey
2
Laboratory of Molecular Biotechnology and Enzymology, Faculty of Science, Department of
Biology, Anadolu University, 26470, Eskişehir, Turkey.
Eukaryotic cells possess myriad adaptations against stress conditions. Among these, autophagy,
a catabolic lysosomal pathway, serves as a turnover mechanism to recycle the redundant and/or
damaged macromolecules present in the cell to re-use them under starvation condition via a
special organelle called autophagosome. Destined-to-degrade cellular components are enclosed
by autophagosome and then fuse with lysosome to form autophagolysosomes. Hydrolytic
enzymes present in the lysosome degrade the cargo material to monomers used again by cell. A
set of eukaryotic genes called autophagy-related genes (ATGs) are known to orchestrate this
highly elaborative process. Existence of these genes and their roles in higher eukaryotes are well
characterized, however, only little is known in lower eukaryotes like ciliates. These eukaryotic
microbes are best characterized by their binucleated genome architecture, a situation known as
nuclear dimorphism. There are two types of morphologically and genetically different nuclei in
the same cytoplasm. Polyploid macronucleus (MAC) governs the cell phenotype by providing all
necessary transcripts for biological functions and serves as soma. Diploid micronucleus (MIC) is
transcriptionally inert and functions as a germline. Both MIC and MAC develops from the zygotic
nucleus during conjugation. Three of four meiotic products and parental MAC are degraded and
eliminated from the cytoplasm in an apoptotic and autophagic-fashion by a genetically and
developmentally programmed process, known as Programmed Nuclear Death (PND). In this
study, we performed extensive in-silico analyses in five model ciliate genomes to understand the
diversification of Atg proteins in lower taxa and discuss their possible roles during PND. Contrary
to previous reports, we found that ciliate genomes do not have typical Atg1 proteins since all the
reported or newly defined candidate sequences are lack of Atg1-specific C-terminal domain,
which is essential for the Atg1 complex formation. We also noticed that several genes in Oxytricha
trifallax and Stylonychia lemnae genomes were annotated as ATG16L, a mammalian version of
yeast ATG16. However, executed domain analysis showed that these proteins do not contain
Atg16 domain. Surprisingly, we failed to find any ATG8 gene, which encodes a ubiquitin-like
protein that is critical for autophagosome formation, in parasitic ciliate Ichthyophthirius multifiliis
while other four ciliates have varying numbers. On the other hand, phylogenetically ATG8-related
gene, ATG12, is only found in O.trifallax. Phylogenetic analysis suggested that Atg8 proteins in
ciliates have evolved to function differently. In addition, we also realized that several non-yeast
mammalian autophagy genes like UVRAG (in O.trifallax) and VMP1 (in T.thermophila) are found
in ciliates. Lastly, we analyzed mRNA expression level of ATGs in T.thermophila and Paramecium
tetraurelia transcriptome databases. ATG4 and ATG8 expressions in both species show significant
changes during conjugation and autogamy respectively, which may further imply a possible
conserved roles during PND. In conclusion, we survey the bioinformatics analyses of autophagy
proteins in model ciliates in the perspective of PND. Understanding the role of autophagy proteins
in lower eukaryotes like ciliates may help to decipher novel functions of these proteins.
Keywords: Ciliate, autophagy-related genes, in silico, autophagy