Amyloids are fibrillar, highly ordered protein aggregates with a typical cross β-sheet structure 
. They appear in cells as well as in the extracellular space and are often associated with protein misfolding disorders, including neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and prion disease 
. Functionally, amyloids are involved in numerous physiological processes such as information transfer in neurons, melanosome biogenesis or modulation of translation termination 
. Although pathogenic and non-pathogenic amyloid assembly are pervasive biological processes, we still lack a comprehensive understanding of the molecular mechanisms that lead to the spontaneous formation of amyloid.
There are striking similarities in the aggregation behaviour of different amyloidogenic peptides and proteins 
. In the initial phase of amyloidogenesis, aggregation-prone monomers relatively slowly form soluble oligomers 
. These earliest aggregation species, visible by electron and atomic force microscopy, are small bead-like structures, also described as amorphous protein aggregates 
. Amyloidogenic oligomers are biochemically and biophysically not very well defined and are currently thought to cause cellular toxicity in numerous amyloid diseases 
. Over time, they transform into larger aggregate species with a more fibrillar morphology, often termed “protofibrils” 
. Protofibrils are better defined with regard to size and biological activity and are also highly toxic for mammalian cells 
. Finally, protofibrils self-assemble efficiently into large mature amyloid fibrils, perhaps through association of monomers or oligomers, which is often accompanied by a structural reorganisation of aggregates 
Currently, the molecular mechanisms behind the initiation of amyloid self-assembly are largely unclear. On the one hand, it is thought that oligomer formation is driven by relatively unspecific protein-protein interactions between monomers resulting in undefined, disordered structures 
. On the other hand, experimental evidence showed the earliest detectable oligomer species to be fairly distinctive structures, suggesting that they are formed by specific protein-protein interactions 
. In any case, the process is slow and inefficient, with a significant entropic barrier that is mainly controlled by the concentration of monomers 
A wide range of factors has been reported to influence spontaneous amyloid assembly 
. Extrinsic factors are, e.g., the physico-chemical properties of the cellular environment of polypeptide chains (pH, temperature, ionic strength, protein concentration) or molecular chaperones inhibiting or promoting aggregation by directly binding to polypeptide chains 
. Intrinsic factors are properties of polypeptides, such as charge, hydrophobicity, patterns of polar and non-polar amino acids and the ability to adopt specific secondary structures 
. In the case of globular proteins, the propensity to spontaneously form amyloid structures is inversely related to the stability of their native states 
. A large number of proteins that assemble into amyloid, however, are at least partially unfolded under physiological conditions 
Experimental evidence has been provided that a high content of the polar amino acids glutamine (Q) and asparagine (N) leads to an increased tendency in proteins to spontaneously form amyloids, implicated in human neurodegeneration and non-Mendelian inheritance of prions in yeast 
. Several neurodegenerative diseases including Huntington's disease and a variety of spinocerebellar ataxias are caused by a pathogenic expansion of CAG codons in disease genes leading to the production of proteins with elongated polyglutamine (polyQ) tracts. These proteins form inclusion bodies in affected neurons in patient brains that correlate with disease progression and toxicity 
. Pathogenic polyQ tracts in the Huntington's disease protein, huntingtin, have been shown to stimulate the assembly of amyloid fibrils in vitro and in vivo 
, suggesting a function as aggregation-promoting sequences driving amyloidogenesis.
Glutamine/asparagine (Q/N)-rich regions are a characteristic feature of the yeast prion proteins Sup35, Rnq1, Ure2 and Swi1 
. In these proteins, the Q/N-rich sequences, as part of prion domains (PrDs), are critical for spontaneous self-assembly of ordered amyloid fibrils and the appearance of defined yeast phenotypes 
. Previous studies have shown that prions with Q/N-rich regions can facilitate the assembly of polyglutamine aggregates in yeast cells 
. Glutamine-rich sequences exist in a wide range of proteins across numerous species, suggesting that they have important physiological functions 
. This is supported by bioinformatic studies, indicating that glutamine-rich domains in proteins are conserved and can undergo positive selection 
. More specifically, they have been implicated as facilitators of protein complex formation 
, however, their physiological roles are largely unclear.
The structural basis of polyQ-mediated protein aggregation is believed to be the formation of “polar zippers”, in which β-sheets are stabilised by hydrogen bonds between polar amino acids 
. Once monomers are joined by hydrogen bonds and a significant number of stable protein complexes, so-called seeds, have appeared, amyloid fibrils are formed efficiently by a nucleation-dependent process 
. Whether polar zipper formation involving Q/N-rich regions in proteins is indeed critical for the initiation of amyloidogenesis, however, is currently unknown. Interestingly, proteins with aggregation-promoting Q/N-rich domains such as the yeast prion protein Sup35 have a relatively low intrinsic propensity to self-assemble 
. In yeast cells, insoluble Sup35 protein aggregates appear de novo
at very low frequency. Spontaneous formation of Sup35 aggregates occurs efficiently only at high protein concentrations and is greatly facilitated by the presence of preformed Q/N-rich, seeding-competent nuclei derived from other proteins 
. This suggests that the aggregation-prone Q/N-rich PrD in the full-length protein is not sufficient to promote spontaneous Sup35 aggregation.
In this study, we have investigated whether polyQ sequences and their ability to form polar zippers can be employed to initiate the amyloidogenesis of soluble Q/N-rich prion proteins such as Sup35. Fusion proteins of Sup35 or its N-terminal PrD with polyQ tracts of different lengths were produced in vitro and in vivo. Subsequently, the spontaneous formation of stable protein aggregates was analysed using biochemical as well as cell biological methods. Our data indicate that only long, pathogenic polyQ tracts (≥54 glutamines) are potent inducers of Sup35 amyloid polymerisation. PolyQ-stabilised Sup35 amyloids are permanently maintained in yeast cells and form independently of the biological activity of the yeast chaperone Hsp104 or other Q/N-rich yeast prions. Similar results were obtained when polyQ-tagged fusions of the prion protein Rnq1 were systematically analyzed in yeast cells.