Prion-specific Hsp40 function: The role of the auxilin homolog Swa2

Figures & data

FIGURE1. Primary sequence diagrams of Swa2 constructs and chimeras examined for [URE3] propagation. Swa2 mutants were tested using plasmid-shuffling experiments.¹ Domains are denoted using the following notation: CB 1–3, clathrin-binding domains 1–3; UBA, ubiquitin associated domain; TPR, tetratricopeptide repeat domain; J, J domain;³⁷ AuxJ, J-domain of human auxilin; Sis1J, J domain of yeast Sis1. (*) denotes single-point mutations. (+) or (-) denotes maintenance or loss, respectively, of [URE3–1], the specific strong variant of [URE3] used in these studies.

TABLE 1. Homology analysis of the Swa2 TPR domain relative to HOP TPR1 and TPR2A. Swa2 TPR residues aligned against HOP TPR1 and TPR2A residues that form critical intermolecular interactions with their respective EEVD ligands. Interactions were determined from crystal structure⁵³ analyses using the molecular graphics modeling system, PyMOL. Alignments were performed using protein sequence alignment tools LALIGN and CLUSTALW.

Hsp70	Interaction	HOP TPR1	SWA2 TPR	HOP TPR2A	Interaction	Hsp90
D^0	salt bridge	K^8	K^378	K^229	salt bridge	D^0
D^0	ion-dipole	N^43	N^417	N^264	ion-dipole	D^0
D^0	ion-dipole	N^12	→T^382	N^233	ion-dipole	D^0
D^0	salt bridge	K^73	K^468	K^301	salt bridge	D^0
			→K^452	Q^298	ion dipole	D^0
V^−1	hydrophobic	L^15	→F^385
V^−1	hydrophobic	A^46	A^420	A^267	hydrophobic	V^−1
			Y^397	Y^248	hydrophobic	V^−1
E^−2	ion-dipole	K^73	K^468
E^−2	ion-dipole	R^77	R^472	R^305	ion-dipole	E^−2
			→R^475	N^308	ion-dipole	E^−2
E^−3	ion-dipole	R^77	R^472	R^305	ion-dipole	E^−3

FIGURE 2. Residues of Swa2's TPR domain, but not Hsp104's C-terminal DDLD motif, are required for [URE3] propagation. (A) Haploid [URE3] cells of the W303 genetic background expressing His3 in place of genomic Swa2 and bearing the plasmid pRS416–SWA2 were transformed with plasmids (pRS315) expressing Swa2 variant constructs lacking the Swa2 N-terminal domains and containing a single amino acid substitution (K³⁷⁸→A or K⁴⁶⁸→A). Swa2-Δ2–362 and swa2-Δ were used as positive and negative controls for prion maintenance, respectively. Colonies exhibiting leucine prototrophy were placed on media containing 5-Fluoroorotic acid (5-FOA) to counter-select against the URA3-marked SWA2 plasmid. Red/white color phenotype assays are shown for representative transformants (n ≥ 8 for each variant); in this yeast genetic background red pigment accumulates upon [URE3] loss. (B) Haploid W303 [URE3] cells possessing genomic HSP104 were crossed with one of 2 strains expressing Leu2 in place of genomic Hsp104, and expressing either wild-type Hsp104 (pRS313, row 1) or an Hsp104 construct lacking its 4 terminal residues: DDLD (pRS314, rows 2 and 3).²² After passage on media selecting for adenine and leucine prototrophy, diploids were sporulated and subjected to tetrad dissection. Color phenotype assays are shown for representative haploids (right columns, n ≥ 7). Haploids were subsequently treated with GdnHCl to inhibit Hsp104 function (left columns, n ≥ 7 for each variant), to confirm that colony color still accurately reported prion status.

FIGURE 3. Proposed model of Cpr7-Hsp90-Swa2 cooperation and complex formation in [URE3] prion fragmentation. Hsp70 is recruited to [URE3] aggregates by formation of a chaperone bridge. Cpr7, bound to the aggregate, complexes with the EEVD of one Hsp90 monomer at its TPR domain while Swa2, bound to Hsp70 at its J-domain, complexes with the EEVD of the other Hsp90 monomer via the Swa2 TPR domain. This multi-chaperone protein complex allows Hsp70 to productively recruit Hsp104 and enhance prion fragmentation.

Troisi EM, Rockman ME, Nguyen PP, Oliver EE, Hines JK. Swa2, the yeast homolog of mammalian auxilin, is specifically required for the propagation of the prion variant [URE3-1]. Mol Microbiol 2015; 97:926-41; PMID:26031938; https://doi.org/10.1111/mmi.13076

 PubMed Web of Science ®Google Scholar

Xiao J, Kim LS, Graham TR. Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo. Mol Biol Cell 2006; 17:3281-90; PMID:16687570; https://doi.org/10.1091/mbc.E06-02-0106

 PubMed Web of Science ®Google Scholar

Scheufler C, Brinker A, Bourenkov G, Pegoraro S, Moroder L, Bartunik H, Hartl FU, Moarefi I. Structure of TPR Domain–Peptide complexes. Cell 2000; 101:199-210; PMID:10786835; https://doi.org/10.1016/S0092-8674(00)80830-2

 PubMed Web of Science ®Google Scholar

Reidy M, Masison DC. Sti1 regulation of Hsp70 and Hsp90 is critical for curing of Saccharomyces cerevisiae [PSI+] prions by Hsp104. Mol Cell Biol 2010; 30:3542-52; PMID:20479121; https://doi.org/10.1128/MCB.01292-09

 PubMed Web of Science ®Google Scholar