A Systematic Evaluation of the Function of the Protein-Remodeling Factor Hsp104 in [PSI⁺] Prion Propagation in S. cerevisiae by Comprehensive Chromosomal Mutations

Figures & data

Figure 1 Hsp104 primary structure and location of mutations engineered in this study. The multi-domain structure of the Hsp104 monomer. The N-terminal domain (NTD), nucleotide-binding domains (NBD) 1 and 2, D1 small domain (D1sD), middle region (MR), and C-terminal domain (CTD) are indicated. The domain boundaries are according to Hsp104's alignment with the T. thermophilus ClpB sequence.6 The hsp104 mutations are shown with their allele numbers, for example, A201V is an Ala-to-Val change at position 201.

Figure 2 The status of [PSI⁺] in hsp104 mutants is monitored by color assay of ade1-14 nonsense suppression. Haploid strains carrying the indicated hsp104 mutations and control wild-type [PSI⁺] strain (AT3) were incubated on YPD at 30°C for four days. The results are summarized in Table 3.

Figure 3 Hsp104-levels in mutant strains. Wild-type [psi^-] (AT1), wild-type [PSI⁺] (AT3), Δhsp104 and 58 missense hsp104 mutants were cultured in YPD+ade liquid medium and harvested in mid-log phase. (Upper) Anti-Hsp104 immunoblotting. (Lower) Coomassie stained internal loading control.

Figure 4 Tetrad analysis of hsp104 mutant phenotypes monitored by nonsense suppression of the ade1-14 strain. Heterozygous diploids from matings between indicated hsp104 mutants and wild-type [PSI⁺] cells (NPK151) were sporulated and their progeny were incubated on YPD at 30°C for four days. Asterisks indicate the Leu⁺, hsp104 mutants.

Figure 5 Effect of hsp104 mutations on thermotolerance. Mid-log phase cultures of the indicated strains were incubated at 37°C for one hour to induce the heat-shock response and were then incubated for a further 20 minutes at 50°C. The viability of heat-shocked cells (right) were compared to untreated cells (left) by five-fold serial dilutions on YPD+ade agar. Cells were incubated at 30°C for two days. The results are summarized in (Table 3).

Figure 6 Structure of the ClpB/Hsp104 monomer and hexamer. (A) Ribbon drawing of T. thermophilus ClpB monomer (Protein Data Bank accession number 1QVR).6 The figure shown was generated with the program PyMOL. Domain assignments were given by Lee et al.6 Amino acid changes in hsp104 mutants (shown as grey, red or blue spheres) are assigned at the equivalent positions in ClpB (shown in parentheses). Red spheres show [PSI⁺]-eliminating thermotolerant mutations. Y257A and A298V mutations are not shown because these positions were not determined in the 3D structure of T. thermophilus ClpB. Blue spheres show hsp104 mutations that were able to propagate [PSI⁺] but lost thermotolerance. (B) Axial (left-top) and lateral (left-bottom) images of ClpB/Hsp104 hexamer (reprinted from Lee et al6 with permission from Elsevier). (Right) A close-up view of the lateral channel. The figure shown was generated with the program PyMOL. D184Y, A201V, T317A, L462R, P557L, D704N and T726I mutations, which remained thermotolerant but lost [PSI⁺] propagation, are indicated as red spheres. Parentheses indicate the equivalent positions in ClpB.

Table 1 Strains

Name	Plasmotype	genotype
AT1	[psi^-]	MATa ade1-14 leu2Δ0 ura3-197 (Trp197-to-UGA) his3Δ200 trp1-289
AT3	[PSI^+]	MATa ade1-14 leu2Δ0 ura3-197 his3Δ200 trp1-289
NPK151	[PSI^+]	MATα ade1-14 leu2-3,112 ura3-52 trp1-289
NPK175	[psi^-]	MATα ade1-14 leu2-3,112 ura3-52 trp1-289

Table 2 Primers used in this study

corresponding hsp104 mutation	Sequence (5′-to-3′)	Corresponding hsp104 mutation	Sequence (5′-to-33′)
T8A	GCCCTTTCTGCAAATTGCGTTTG	A551T	CTGAAACAGCTACAAGATTGACTG
L15Q	GAGCCAACGTCTGAATCGTTAGAGC	V558I	CTGGTATCCCTATTAAGAAGTTGTC
K20E	GTTGGCTCAAGAATTGGCTTCG	S597P	CCGTTAGATTGCCTAGATCAGG
A22V	CTCAAAAATTGGTTTCGGATCATC	G619V	TCCGGTTCCGTTAAAACTGAATTG
S89A	CCAAAGCATAAGCTGGAGTTATCTC	K620T	CGGTTCCGGTACAACTGAATTGG
L96Q	GCAGCGTCTTGCTGGACTTTCCC	E645K	CTCGCTTAATTTAGAACAATCGAC
D108A	GCTATAAATGAGGCCTTCTGTTG	Y662A	CCACGGCAGGTGCTGTCGGGTACG
D184N	CGATGACAGGGTTAAGTTTACCTTG	P679L	GCAATACAAACTATACTCCGTTTTG
D184Y	CGATGACAGGGTAAAGTTTACCTTG	E687A	GTTATTCGATGCAGTAGAAAAGGC
A201V	TAGAGTTTTAGTAAGAAGAATTAAG	I722T	TGTTCCAATTGTACTGTCATCATGACT
G217V	CCAGGTATCGTTAAGACCGCT	T726I	GTCATCATGATTTCCAATCTAGG
K218T	TAATAGCGGTCGTACCGATACCT	N728A	CATGACTTCCGCTCTAGGTGCTG
Y257A	ATCACCTTTGGCTTTAGCACCTGCG	S745P	CAAATTCTTGGTAGGTTCTTGGATC
E285A	CATGTGAATTGCATCAATGAATAA	K747R	CCATGACCAAATTCCTGGTAGATTC
A298V	GGACGACGCTGTTAACATCTTG	R765A	ACTAGAAATTGCGTTCAAAAATTCTGG
T317A	CGGTGCCACCGCCAATAACGAAT	F772S	GATAGCTTGTTGGAAATGACTATAC
A341V	ATTGAAGTCGTTGAACCAAGTG	K774E	TCTAGATAGCTCGTTGAAAATGAC
G364N	GAAATACATCATAATGTAAGGATTC	L775P	GCTTTTCTAGATGGCTTGTTGAA
V365I	ACATCATGGTATAAGGATTCTGG	L814S	CATATTTGGCCGAGAAGTCCTTGG
A392T	GCCAGATTCTACTTTGGATTTAG	R826M	GATATGGGTGCAATGCCACTGAACAGG
A430V	GTAGAGATAAAAGTTCTAGAGAGAG	R830A	TTGAATTAACGCGTTCAGTGGTC
T499I	GTAGATATGATATTGCTACCGCC	L840Q	TTGAACAAACAGGCACTAAGG
A503V	CTGCTACCGCCGTTGATTTAAGGT	L845I	GCACTAAGGATCATAAAGAATGA
A509D	AGGTACTTCGACATCCCAGATATC	E878G	GAATGTCTGGGAGTTCTACCA
A531V	GAGAGACGTGTTGGTGCCAAC

Table 3 Properties of hsp104 mutations

Domain	Mutation	[PSI^+] Propagationa	Thermotoleranceb	conservation (%)c	Noted	Ref. No.
	T8A	±	+	60	N	35
	L15Q	+	+	80	N	36
N-terminal	K20E	+	+	20	M	29
domain	A22V	+	+	80	M	29
	S89A	+	+	40	N	35
	L96Q	+	+	60	N	36
	D108A	±	+	40	N	35
	D184N	-	±	100	B	30
	D184Y	-	+	100	B	30
	A201V	-	+	0	M	29
	C209Y	-	±	100	N	27
	G212D	-	-	100	N	27
	G217V	-	-	100	T	38
NBD1	K218T	-	-	100	B	22,34
	A220T	-	±	100	N	27
	Y257A	-	+	100	T	7
	G259D	-	-	100	N	27
	E285A	-	-	100	N	31
	A298V	-	+	40	M	29
	T317A	-	+	100	B	32
	A341V	+	+	40	M	29
	G364N	+	+	60	M	29
D1-small	V365I	+	+	100	M	29
domain	P389L	-	±	100	N	27
	A392T	±	±	100	M	29
Middle	A430V	±	+	60	M	29
region	L462R	-	+	80	N	27
	T499I	-	±	0	M	29
	A503V	±	+	40	T	29,39
	A509D	-	-	20	T	29
	A531V	+	+	0	M	29
D1-small domain	A551T	+	+	60	M	29
	P557L	-	+	100	N	27
	V558I	+	+	60	M	29
	S597P	±	±	40	M	29
	G619V	-	-	100	T	38
	K620T	-	-	100	B	22,34
	E645K	-	-	100	T	7
	G661D	-	-	100	N	27
	Y662A	-	-	100	T	7
	P679L	-	±	100	M	29
NBD2	E687A	-	-	100	N	31
	D704N	-	+	100	N	27
	I722T	+	+	20	T	28
	T726I	-	+	100	M	29
	N728A	-	-	100	B	32
	S745P	+	+	0	M	28
	K747R	+	+	40	M	28
	R765A	-	-	100	N	31
	F772S	±	±	100	B	28
	K774E	±	±	0	B	28
	L775P	±	±	100	M	28
	L814S	+	-	80	B	28
C-terminal	R826M	-	±	100	B	33
domain	R830A	-	±	100	N	37
	L840Q	+	±	80	B	28
	L845I	+	+	80	M	28
	E878G	+	+	20	M	28

a +, normal [PSI⁺] propagation; -, loss-of-[PSI⁺]; ±, weak [PSI⁺] propagation

b The assessments were evaluated by three independent experiments. -, loss-of thermotolerance; ±, weak thermotolerance; +, normal thermotolerance.

c The frequency of amino acid conservation at the indicated positions in S. cerevisiae Hsp104 relative to T. thermophilus ClpB, E. coli ClpA and ClpB, A. thaliana Hsp101 and S. cerevisiae Hsp78. Alignments refer to Lee et al.6

d History about each mutations: N, newly engineered in this laboratory including those of very recent publication;27 M, mutations reported in the literature as multiple amino acid substitutions and each mutation not clarified independently; T, mutations known to affect only thermotolerance; B, mutations known to affect both thermotolerance and [PSI⁺].

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