Aggregation and Prion-Inducing Properties of the G-Protein Gamma Subunit Ste18 are Regulated by Membrane Association

Abstract

Yeast prions and mnemons are respectively transmissible and non-transmissible self-perpetuating protein assemblies, frequently based on cross-β ordered detergent-resistant aggregates (amyloids). Prions cause devastating diseases in mammals and control heritable traits in yeast. It was shown that the de novo formation of the prion form [PSI+] of yeast release factor Sup35 is facilitated by aggregates of other proteins. Here we explore the mechanism of the promotion of [PSI+] formation by Ste18, an evolutionarily conserved gamma subunit of a G-protein coupled receptor, a key player in responses to extracellular stimuli. Ste18 forms detergent-resistant aggregates, some of which are colocalized with de novo generated Sup35 aggregates. Membrane association of Ste18 is required for both Ste18 aggregation and [PSI+] induction, while functional interactions involved in signal transduction are not essential for these processes. This emphasizes the significance of a specific location for the nucleation of protein aggregation. In contrast to typical prions, Ste18 aggregates do not show a pattern of heritability. Our finding that Ste18 levels are regulated by the ubiquitin-proteasome system, in conjunction with the previously reported increase in Ste18 levels upon the exposure to mating pheromone, suggests that the concentration-dependent Ste18 aggregation may mediate a mnemon-like response to physiological stimuli.

Yeast Chaperone Hsp70-Ssb Modulates a Variety of Protein-Based Heritable Elements

“…In the WT strain, Lsb2 promoted [PSI + ] induction after both simultaneous and sequential overexpression, while Ste18 cross-seeded [PSI + ] only in the simultaneous and not the sequential protocol (Figure 6A). This result agreed with our previous data [13] and was expected as aggregates of Ste18 are entirely non-heritable in the WT strain in contrast to aggregates of Lsb2 which are heritable. However, we found that overexpressed Ste18 is able to promote [PSI + ] formation both after simultaneous and after sequential overproduction in the ssb1/2 background (Figure 6A).…”

“…Another yeast protein, a Gγ-subunit (Ste18), forms non-heritable detergent-resistant aggregates upon overproduction in the WT strain [13]. These aggregates resemble "mnemons" previously described for another yeast protein in the pheromone signaling pathway, Whi3 [12].…”

“…The yeast Ste18 protein is a γ-subunit of G-protein, which is involved in the pheromone signaling pathway and is shown by us [13] to form a non-heritable amyloid-like aggregate with properties of yeast mnemon upon overproduction. Similar to Lsb2, aggregated Ste18 can cross-seed Sup35 into a prion form, [PSI + ].…”

“…Mnemon formed by the Whi3 protein is responsible for the memory of failed mating [12]. We have previously demonstrated that a detergent-resistant aggregate of Ste18, the γ subunit of the heterotrimeric G-protein complex, which is also involved in the mating pathway, could be induced by protein overproduction and behaves as a non-heritable mnemon [13].…”

N-terminal intrinsic disorder is an ancestral feature of Gγ subunits that influences the balance between different Gβγ signaling axes in yeast

“…S3 ). We also could not find evidence that these mutations resulted in abnormal aggregation of Ste18, as has been recently reported under conditions of extraordinarily high cell density ( 42 ). Surprisingly, comparative analysis of predicted mRNA secondary structure does suggest the potential for unique structures to form for M2–M4 that are adjacent to the translation start site, which we speculate could disrupt translation initiation of these mRNAs ( Fig.…”