Identification of the amyloidogenic domain in the integrase of yeast retrotransposon Ty1

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Abstract

BACKGROUND: Retrotransposons are mobile genetic elements that replicate via reverse transcription and constitute substantial fractions of eukaryotic genomes; they are also considered evolutionary precursors of retroviruses, and can affect host fitness. Intriguingly, some retrotransposon proteins share motifs with amyloids—fibrous protein aggregates with cross-β architecture that readily self-assemble into polymeric structures and can, in some cases, self-propagate in an infectious manner (prions).

AIM: To identify and characterize potential amyloid-forming regions within the integrase of Saccharomyces cerevisiae Ty1 retrotransposon, which mediates integration of transposon copies into the host genome.

METHODS: Computational analysis of the Ty1 integrase sequence was performed with the ArchCandy algorithm to identify putative amyloidogenic motifs. To evaluate the amyloidogenic potential of candidate regions, we employed a yeast-based nucleation assay. Aggregation was visualized by expressing Ty1Int(AD)-GFP fusion constructs. Colocalization of Ty1 amyloidogenic domain with full length in yeast cells was evaluated by confocal microscopy.

RESULTS: We identified and experimentally validated an amyloidogenic region within the Ty1 integrase, designated Ty1Int(AD). ArchCandy predicted the region with amyloidogenic potential, and these predictions were confirmed in a yeast prion-nucleation assay and by expression of the Ty1Int(AD)-GFP fragment, which formed detergent-resistant aggregates. Confocal microscopy showed co-localization of these aggregates with native Ty1 integrase fused with YFP, indicating recruitment of the full-length protein into inclusions.

CONCLUSION: Together, these results identify a previously unrecognized amyloidogenic region within Ty1 integrase, possessing amyloid-like properties, and suggest that aggregation of this domain may regulate retrotransposon activity by altering integrase availability and/or function. This is therefore important for the design, optimization, and biosafety assessment of retrotransposon based vectors and other GMO constructs.

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About the authors

Andrew A. Zelinsky

Saint Petersburg State University

Email: andrew_zelinsky@mail.ru
ORCID iD: 0000-0003-2068-3024
SPIN-code: 5832-1192
Russian Federation, Saint Petersburg

Marina V. Ryabinina

Saint Petersburg State University

Email: marina.v1205@gmail.com
ORCID iD: 0000-0002-5504-7362
SPIN-code: 7113-6941
Russian Federation, Saint Petersburg

Andrey V. Kajava

Saint Petersburg State University; University of Montpellier

Email: andrey.kajava@crbm.cnrs.fr
ORCID iD: 0000-0002-2342-6886
SPIN-code: 5631-3525

Cand. Sci. (Biology), Professor

Russian Federation, Saint Petersburg; France, Montpellier

Yury O. Chernoff

Georgia Institute of Technology

Email: yury.chernoff@biology.gatech.edu
ORCID iD: 0000-0002-8934-9051
SPIN-code: 6201-0359

PhD, Professor, School of Biological Sciences

United States, Atlanta

Aleksandr A. Rubel

Saint Petersburg State University

Author for correspondence.
Email: a.rubel@spbu.ru
ORCID iD: 0000-0001-6203-2006
SPIN-code: 3961-4690

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

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Supplementary files

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2. Fig. 1. In silico predictions: a, Ty1 retrotransposon polyprotein architecture and amyloid forming domain predictions obtained with ArchCandy; b, Structure and functional domain organization of the Ty1 integrase predictions obtained with ArchCandy. Ty1Int(AD) (residues 295–322). The N-terminal domain (NTD; residues 10–64); the catalytic core domain (CCD; residues 75–240); the C-terminal domain (CTD) homologous to retroviral CTD (residues 260–306); the extended C-terminal domain (eCTD, residues 307–375) and disordered C-terminal domain (dCTD, residues 376–635) defined in [23]; nuclear localization signal and targeting domain (NLS+TD, residues 596–630) important for nuclear import and interaction with RNA Polymerase III, respectively; c, ADOPT disorder prediction profile indicating structured regions (residues 1–375) and a transition to disorder at residue ~376 onward.

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3. Fig. 2. Amyloid-like properties of Ty1int(AD) in yeast assays: a, a schematic workflow a yeast-based prion nucleation assay. The candidate sequence is fused to the N-terminal prion domain of the translation termination factor Sup35 (Sup35N). In [pin−] cells Sup35N itself, or when attached to a non-amyloidogenic partner, cannot efficiently initiate prion formation. An amyloidogenic protein, however, drives Sup35N polymerization, converting it to the [PSI+] prion and sequestering full length Sup35 into insoluble aggregates. Sequestration and partial inactivation of full-length Sup35 causes readthrough of the ade1-14 (UGA) nonsense mutation, allowing growth on adenine-dropout selective medium (−Ade); b, analysis of Sup35-Ty1int(AD)-mediated nonsense suppression in S. cerevisiae. Growth colonies after 15 days of cultivation at 30 °C on adenine-free selective medium. GT409 [psi–][pin–] was used as the host strain for analysis of the experimental plasmid and for the negative control. GT159 [psi–][PIN⁺] strain was used for the positive control. Transformant growth following PCUP1 promoter-driven induction is shown on selective media containing 50, 100, and 150 μM CuSO4 as well as on medium without added CuSO4; c, SDD AGE of detergent resistant aggregates formed by Ty1Int(AD)-GFP (lane 2) and full-length Ty1Int-YFP (lane 4). Blots were probed with an anti-GFP antibody (cross-reactive with YFP and CFP). Sup35N-GFP expressed in strains GT159 (lane 1) and GT409 (lane 3) were included as positive and negative controls, respectively. Samples were treated with 3% sodium lauroyl sarcosinate (final concentration) prior to electrophoresis. Representative of 3 independent experiments; d, fluorescence images of Ty1Int(AD)-GFP and the negative control Sup35N-GFP expressed in strain GT409; Sup35N-GFP expressed in strain GT159 served as the positive-control strain. Scale bar = 10 µm.

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4. Fig. 3. Representative fluorescence micrographs showing colocalization of protein aggregates formed by Ty1Int(AD) and full-length integrase in [psi–][pin–] yeast cells. Left to right: CFP channel (Ty1Int(AD) signal), YFP channel (Ty1Int signal), bright-field (BF), and merged overlay of all channels. Images are single optical sections acquired by confocal fluorescence microscopy. Scale bar = 10 µm.

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