Amyloid properties of the yeast cell wall protein Toh1 and its interaction with prion proteins Rnq1 and Sup35

Abstract

Amyloids are non-branching fibrils that are composed of stacked monomers stabilized by intermolecular β-sheets. Some amyloids are associated with incurable diseases, whereas others, functional amyloids, regulate different vital processes. The prevalence and significance of functional amyloids in wildlife are still poorly understood. In recent years, by applying new approach of large-scale proteome screening, a number of novel candidate amyloids were identified in the yeast Saccharomyces cerevisiae, many of which are localized in the yeast cell wall. In this work, we showed that one of these proteins, Toh1, possess amyloid properties. The Toh1-YFP hybrid protein forms detergent-resistant aggregates in the yeast cells while being expressed under its own P TOH1 or inducible P CUP1 promoter. Using bacterial system for generation of extracellular amyloid aggregates C-DAG, we demonstrated that the N-terminal Toh1 fragment, containing amyloidogenic regions predicted in silico, binds Congo Red dye, manifests 'apple-green' birefringence when examined between crossed polarizers, and forms amyloid-like fibrillar aggregates visualized by TEM. We have established that the Toh1(20-365)-YFP hybrid protein fluorescent aggregates are co-localized with a high frequency with Rnq1C-CFP and Sup35NM-CFP aggregates in the yeast cells containing [PIN + ] and [PSI + ] prions, and physical interaction of these aggregated proteins was confirmed by FRET. This is one of a few known cases of physical interaction of non-Q/N-rich amyloid-like protein and Q/N-rich amyloids, suggesting that interaction of different amyloid proteins may be determined not only by similarity of their primary structures but also by similarity of their secondary structures and of conformational folds.

Functional Mammalian Amyloids and Amyloid-Like Proteins

“…PSIA method was applied to identification of new amyloid proteins (potential functional amyloids) in bacteria [ 236 , 237 ] and yeast [ 238 ], as well as in the rat hippocampus [ 120 ]. Amyloidogenic properties of some of these proteins, including bacterial proteins YghJ, RopA and RopB, yeast proteins Gas1, Toh1 and Ygp1, and the abovementioned rat Fxr1 have been then confirmed by using a variety of standard amyloid characterization techniques [ 117 , 237 , 238 , 239 , 240 ]. An approach similar to PSIA has been independently developed by F. Shewmaker lab and applied to identification of yeast prions [ 241 ].…”

“…This approach was successfully tested using several known yeast amyloidogenic proteins (Sup35NM, Rnq1, Cyc8, and New1) and polyQ (aggregating) region of human huntingtin (Htt72Q), while the non-aggregating linker domain of yeast Sup35 protein (Sup35M) and non-aggregating derivative of human huntingtin (Htt25Q) were used as negative controls [ 242 ]. At subsequent stages, the C-DAG system was used to confirm the amyloidogenic properties of several yeast proteins (Mss11 and Pub1 [ 242 ], Gas1 and Ygp1 [ 238 ], and Toh1 [ 240 ]), some bacterial proteins (biofilm-associated proteins especially from Enterococcus faecalis and Bap from S. aureus [ 244 ], RopA and RopB from Rhizobium leguminosarum [ 237 ], and YghJ from E.coli [ 239 ]), and Fxr1 protein from rat Rattus norvegicus [ 120 ]. C-DAG approach was suggested as a convenient method for screening of potentially amyloidogenic proteins from DNA libraries [ 242 ].…”

Single-molecule studies of amyloid proteins: from biophysical properties to diagnostic perspectives

“…Zadorsky's group used FRET to demonstrate that the newly found amyloid protein Toh1, which is localized in the yeast cell wall, could interact with other PrPs Rnq1 and Sup35. The result highlighted that the interactions among different amyloid proteins not only occur between molecules with a similar primary structure but also between proteins that have a close secondary structure and conformational folds (Sergeeva et al ., 2019).…”

FRET Microscopy in Yeast

“…Finally, FRET has also been used to map interactions of heterologous proteins expressed in yeast. Very interesting examples of this approach are studies on disease-related aggregation of Prp prion alone or with amyloid β peptide [38,39], hungtingtin protein [40], or yeast protein Toh1 with yeast prions Rnq1 and Sup35 [41]. The protein complexes mapped by FRET in yeast are summarized in Table 1, also highlighting the used FRET fluorophores and applied techniques.…”

“…Nuclear pore complex (NPC) sensitized emission CFP-YFP [11,12,42] Spindle pole body (SPB) sensitized emission acceptor photobleaching CFP-YFP mTq2-YFP [16][17][18][19][20] Kinetochore sensitized emission FLIM GFP-mCherry mTq2-YFP [13][14][15] Contractile ring acceptor photobleaching GFP/mNG-mCherry [21] Endocytic coat acceptor photobleaching GFP-mCherry mTq-mNG mNG-mScarlet [22] Cohesin sensitized emission CFP-YFP [24] SAGA-Gal4 transcription factor acceptor photobleaching spectral FRET CFP-YFP [25,43] PCNA-SAS-I complex FLIM CFP-YFP [26] ATR complex Dcp2-Mec1-PP4 phosphatase Psy2-Php3 sensitized emission GFP-RFP [27] Ste2 oligomerization spectral FRET CFP/GFP-YFP [28][29][30]44] Fet3-Ftr1 iron permease spectral FRET CFP-YFP [31] Ctr1 transporter oligomerization and copper binding spectral FRET CFP-YFP [32] Ato1-Ato2 proteins acceptor photobleaching, FLIM GFP-tdimer2 CFP-Venus [33] V-ATPase disassembly sensitized emission CFP-YFP [34] Tom70 oligomerization sensitized emission CFP-YFP [35] CDK inhibitor Sic1-cyclins FLIM mCerulean-YFP [36] Ste5-Fus3 interaction acceptor photobleaching GFP-mStrawberry [37] Prp prion aggregation donor photobleaching CFP-YFP [38] Prp-amyloid β interaction acceptor photobleaching CFP-YFP [39] HTT huntingtin aggregation acceptor photobleaching CFP-Venus [40] Toh1 aggregation with Rnq1 and Sup35 prion proteins acceptor photobleaching CFP-YFP [41] 1 For individual FRET techniques and fluorescent proteins, see please the main text. 2 FRET-based protein proximity mapping in yeast can also encounter some difficulties.…”