Characterization of Proteins Associated with Polyglutamine Aggregates

Figures & data

Figure 1 Isolation of 103Q aggregates from yeast cells. (A) Fluorescent micrograph (FITC channel) of yeast wild type cells with GFP-labeled 103Q aggregates after 6 h induction. (B) Scheme of the purification procedure. (C) Fluorescent micrographs of the indicated fractions at x100 magnification; a small portion of the immunoprecipitate (IP) was resuspended in the lysis buffer to obtain the image. (D) SDS-PAGE (Coomassie stained) gel shows approximately equal IgG distribution between IP and supernatant and almost complete purification of the IP from BSA which remains the major band in the non-precipitated fraction, but is almost excluded from the precipitate. Precipitates were reconstituted in the SDS-containing buffer in a volume equal to the volume of the supernatant.

Figure 2 Isolated aggregates and control fraction analyzed by 2-D electrophoresis gels stained with Coomassie blue.

Figure 3 Mass spectra from one of the identified proteins. (A) A representative MALDI-TOF mass spectrum shows matched peptides from the identified protein, Sgt2. The sequence of the Sgt2 protein is shown on the right top corner, identified peptides are underlined. Matched peptides and the monoisotopic m/z values of fragments are indicated above each peak. (B) A representative ESI MS/MS spectrum confirms the identification of proteins by tandem MS/MS of [M+2H]²⁺ m/z 697.9 matching peptide m/z 1394.7 of the panel a. Matched peptide sequence of Sgt2 protein is shown on the top and resulting b and y ions are indicated.

Figure 4 Colocalization of the proteins coprecipitated with 103Q aggregates. Fixed yeast cells with GFP-tagged endogenous proteins (marked on the right) and transformed with mRFP-tagged 103Q were grown in selective medium with raffinose and induced for 6 h with galactose.

Figure 5 Isolation and analysis of 103Q aggregates from cells without prions. (A) Morphology of 103Q aggregates formed in hsp104 cells differs significantly from those in wild type cells (Fig. 1A). (B) Fractions after gel filtration of lysates of hsp104 cells expressing 103Q analyzed by immunoblot with anti-GFP antibody, and quantified by Quantity One software (Bio-Rad). (C) Comparison of chaperones in immunoprecipitates from the wild type and mutant cells. Immunoblot with the respective antibodies (indicated on the right). To detect 103Q, true blot rabbit anti-mouse IgG was used as a secondary antibody. The aggregates were isolated from equal volumes of yeast cultures and solubilized in equal volumes of the Laemmli loading buffer.

Table 1 List of identified proteins associated with 103Q aggregates

Class	Spot Number	NCBI Acc. No.	Protein Name	MWa, kDa	PIb	No of Peptides	Sequence Converage (%)
	1	NP_009396	SSA1/YAL005C (Hsp70)	69.77	4.8	16	37
	2	NP_013076	SSA2/YLL024C (Hsp70)	69.62	4.9	8	21
Chaperone	3	NP_010052	SSB1/YDL229W (Hsp70)	66.75	5.3	7	23
	4,5	NP_010456	HSP42/YDR171W	42.79	5.0	11	39
	16,17,18	NP_014391	SIS1/YNL007C (Hsp40)	37.57	9.0	26	52
Cochaperone	9	NP_014649	SGT2/YOR007C	37.26	4.7	16	49
	6,7	NP_012044	ENO2/YHR174W	46.91	5.9	10	27
Glycolytic	8	NP_009938	PGK1/YCR012W	44.60	7.2	8	25
Enzyme	10	NP_012863	FBA1/YKL060C	39.89	5.5	10	55
	11	NP_014555	ADH1/YOL086C	37.26	6.3	7	19
	15	NP_011708	Tdh3/YGR192C(GAPDH)	35.84	6.5	13	53
Signaling	12,13	NP_011104	BMH1/YER177W	30.19	4.8	9	38
Prion	19	NP_015480	PIN3/YPR154W	23.58	6.8	6	25
	20	NP_014191	YNL208W	20.13	7.0	5	17
Unknown	14	NP_009565	IPP1/YBR011C	32.34	5.4	7	23
	21	NP_014480	YOL162W	24.69	9.9	5	19

Spot numbers refers to Figure 2, top panel.

a Theoretical molecular weight.

b Theoretical isoelectric point.

Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM, Stefani M. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 2002; 416:507 - 511

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