Curing of the [URE3] prion by Btn2p, a Batten disease-related protein

Abstract

[URE3] is a prion (infectious protein), a self-propagating amyloid form of Ure2p, a regulator of yeast nitrogen catabolism. We find that overproduction of Btn2p, or its homologue Ypr158 (Cur1p), cures [URE3]. Btn2p is reported to be associated with late endosomes and to affect sorting of several proteins. We find that double deletion of BTN2 and CUR1 stabilizes [URE3] against curing by several agents, produces a remarkable increase in the proportion of strong [URE3] variants arising de novo and an increase in the number of [URE3] prion seeds. Thus, normal levels of Btn2p and Cur1p affect prion generation and propagation. Btn2p-green fluorescent protein (GFP) fusion proteins appear as a single dot located close to the nucleus and the vacuole. During the curing process, those cells having both Ure2p-GFP aggregates and Btn2p-RFP dots display striking colocalization. Btn2p curing requires cell division, and our results suggest that Btn2p is part of a system, reminiscent of the mammalian aggresome, that collects aggregates preventing their efficient distribution to progeny cells.

Figure 1

Overexpression of CUR1 and BTN2 cures [URE3]. (A) Transformants of BY241 [URE3] v1 or [ure-o] (lacking [URE3]) with multicopy plasmids carrying the indicated genes. Controls (C): pRS425 vector; +: centromeric plasmid; ++: high-copy plasmid and native promoters; +++: high-copy plasmid and TEF2 promoter. (B) Expression levels of Hsp70s, Sse1p and Ydj1p are not altered by overproduction of Btn2p or Cur1p. Lysates of BY241 [ure-o] wild-type, btn2Δcur1Δ and overproduced Btn2p, Ydj1p, Sse1p, Cur1p and Ssa1p cells were analysed by immunoblotting with antibodies against Sse1p, Hsp70s or Ydj1p. Act1p detection was used as a loading control.

Figure 2

Deletions of BTN2 or CUR1 do not destabilize [URE3] and lead to increased induction rates for strong variants of [URE3]. (A) BY241 [ure-o] and [URE3] wild-type strains and with the indicated deletions were streaked on ½ YPD. (B) BY241 [ure-o] wild-type and btn2Δcur1Δ cells were transformed with pH376-Ure2. Following the expression of Ure2p, about 10⁶ cells were spread on plates lacking adenine to select [URE3]. Photographs were taken after 8 days of growth.

Figure 3

Cellular localizations of Btn2p and Cur1p. (A) Expression of Btn2–GFP from a single-copy plasmid (pRS316-Btn2-GFP) and from a high-copy plasmid (pH400tef2-Btn2-GFP). (B) Perinuclear localization of Btn2–RFP (expressed from pRS316-Btn2-RFP) is shown by comparison with nucleoplasmic protein GFP–Pus1. (C) The colocalization of GFP–Cur1 and RFP–Pus1 indicates the nuclear localization for Cur1p.

Figure 4

Btn2p is present in high molecular weight complexes and has little effect on the assembly of Ure2p into amyloid fibres. (A) Centrifugation analysis of BY241 [ure-o] carrying centromeric (cen) or high-copy (h.c.) plasmids expressing Btn2–GFP. T, total lysate; S, supernatant fraction; P, pellet fraction; k=1000 g. Fractions were analysed by western blot with anti-GFP antibodies. The distribution of cytosolic proteins, Hsp70s (B) and Act1p (C), was also detected in the same fractions with corresponding antibodies. (D) Kinetics of Ure2p amyloid formation. Thioflavin-T was used as a reporter of Ure2p amyloid formation. Ure2p (10 μM) was incubated with 20 μM GST, GST–Btn2p or Ydj1p, with constant stirring at room temperature. The normalized fluorescence change is presented (λ_ex=420 nm; λ_det=480 nm).

Figure 5

Colocalization of Btn2p with [URE3] prion aggregates during the curing process. (A) BY241 [URE3] v1 was transformed with pVTG12 to decorate prion aggregates and pYES52-Btn2-RFP for Btn2p–RFP induction. Efficient colocalization was observed during 15–24 h of induction and visualized by fluorescence confocal microscopy. (B) Time course of [URE3] curing for the strain from (A) grown in the presence of raffinose, galactose or galactose and alpha factor. (Inset) The expression level of Btn2p–RFP during induction; cen: Btn2p–RFP expressed from pRS316-Btn2-RFP. (C) The same strain was grown in the presence of galactose and alpha factor and subjected to confocal microscopy. ‘Schmoo' morphology was observed after the addition of alpha factor.

Figure 6

Colocalization of Btn2p with [PSI⁺] prion aggregates and polyglutamine aggregates. (A) 5V-H19 [PSI⁺] was transformed with 181-Sup35NM-GFP to decorate prion aggregates and pYES52-Btn2-RFP for Btn2–RFP induction. Efficient colocalization was observed during 15–24 h of induction and visualized by fluorescence confocal microscopy. (B) BY241 [ure-o] cells carrying pRS425-Btn2-RFP and pQ103-GFP were grown in the presence of galactose for 4–6 h to induce Q103–GFP expression. After that, cells were visualized by fluorescence confocal microscopy.

Figure 7

[URE3] cells contain less propagons than [PSI⁺] cells. BY241 [URE3] wild-type or btn2Δcur1Δ as well as 5V-H19 and 74-D694 [PSI⁺] cultures were grown logarithmically in YPD with 3 mM GuHCl. The percentages of prion-positive colonies recovered following plating on ½ YPD have been plotted as a function of generations, which have been calculated from OD₆₀₀ and colony counts. The 63.2% line was used to estimate propagon numbers.

Figure 8

A model for Btn2 function in managing of prion aggregates. (A) Wild-type cells: prion aggregates are normally distributed to progeny cells during cytokinesis. Btn2p interacts with some prion seeds, reducing their quantity. (B) Cells with overproduced Btn2p: Btn2p sequesters prion aggregates and prevents their efficient distribution to progeny cells. In addition, it may target prion aggregates to vacuolar degradation.