Rsp5/​Nedd4 is the main ubiquitin ligase that targets cytosolic misfolded proteins following heat stress

The heat-shock response is a complex cellular program that induces major changes in protein translation, folding and degradation to alleviate toxicity caused by protein misfolding. Although heat shock has been widely used to study proteostasis, it remained unclear how misfolded proteins are targeted for proteolysis in these conditions. We found that ​Rsp5 and its mammalian homologue ​Nedd4 are important E3 ligases responsible for the increased ubiquitylation induced by heat stress. We determined that ​Rsp5 ubiquitylates mainly cytosolic misfolded proteins upon heat shock for proteasome degradation. We found that ubiquitylation of heat-induced substrates requires the Hsp40 co-chaperone ​Ydj1 that is further associated with ​Rsp5 upon heat shock. In addition, ubiquitylation is also promoted by PY ​Rsp5-binding motifs found primarily in the structured regions of stress-induced substrates, which can act as heat-induced degrons. Our results support a bipartite recognition mechanism combining direct and chaperone-dependent ubiquitylation of misfolded cytosolic proteins by ​Rsp5

Lack of developmental redundancy between Unc45 proteins in zebrafish muscle development.

Since the majority of protein-coding genes in vertebrates have intra-genomic homologues, it has been difficult to eliminate the potential of functional redundancy from analyses of mutant phenotypes, whether produced by genetic lesion or transient knockdown. Further complicating these analyses, not all gene products have activities that can be assayed in vitro, where the efficiency of the various family members can be compared against constant substrates. Two vertebrate UNC-45 homologues, unc45a and unc45b, affect distinct stages of muscle differentiation when knocked down in cell culture and are functionally redundant in vitro. UNC-45 proteins are members of the UCS (UNC-45/CRO1/She4p) protein family that has been shown to regulate myosin-dependent functions from fungi to vertebrates through direct interaction with the myosin motor domain. To test whether the same functional relationship exists between these unc45 paralogs in vivo, we examined the developmental phenotypes of doubly homozygous unc45b(-/-); unc45a(-/-) mutant zebrafish embryos. We focused specifically on the combined effects on morphology and gene expression resulting from the zygotic lack of both paralogs. We found that unc45b(-/-) and unc45b(-/-); unc45a(-/-) embryos were phenotypically indistinguishable with both mutants displaying identical cardiac, skeletal muscle, and jaw defects. We also found no evidence to support a role for zygotic Unc45a function in myoblast differentiation. In contrast to previous in vitro work, this rules out a model of functional redundancy between Unc45a and Unc45b in vivo. Instead, our phylogenetic and phenotypic analyses provide evidence for the role of functional divergence in the evolution of the UCS protein family

Myosin localization in the craniofacial muscles of 4 dpf embryos.

<p>Wild type siblings (a,a'); <i>unc45a^−/−</i> (b,b'); <i>unc45b^−/−</i> (c,c'); <i>unc45b^−/−</i>; <i>unc45a^−/−</i> (d,d') mutants. Lateral (a–d) and ventral (a'–d') views, head to the left. Expression is unaltered between wild type siblings (a,a') and <i>unc45a^−/−</i> (b,b'), <i>unc45b^−/−</i> (c,c') or <i>unc45b^−/−</i>; <i>unc45a^−/−</i> (d,d') mutants. The sternohyoideus is displaced in <i>unc45b^−/−</i> (c,c') and <i>unc45b^−/−</i>; <i>unc45a^−/−</i> (d,d') embryos. Primes denote alternative views of the same embryo. ao, adductor operculi; am, adductor mandibulae; do, dilator operculi; dpw 1–5, dorsal pharyngeal wall; hh, hyohyoideus; ih, interhyoideus; ima, intermandibularis anterior; imp, intermandibularis posterior; lap, levator arcus palatini; pp, pterygoid process; sh, sternohyoideus.</p

<i>hsp90a</i> mRNA is up-regulated in <i>unc45b^−/−</i> mutant embryos at 48 hpf.

<p><i>In situ</i> hybridization with anti-sense probes for <i>hsp90a.1</i> (a,d,g,j), <i>hsp90a.2</i> (b,e,h,k), and <i>hsp90ab.1</i> (c,f,i,l) mRNA transcripts was performed on wild type siblings (a–c); <i>unc45a^−/−</i> (d–f); <i>unc45b^−/−</i> (g–i); and <i>unc45b^−/−</i>; <i>unc45a^−/−</i> (j–l) mutants. <i>unc45b^−/−</i> and <i>unc45b^−/−</i>; <i>unc45a^−/−</i> mutants displayed increased expression of <i>hsp90a.1</i> (g,j) and <i>hsp90a.2</i> (h,k), but not <i>hsp90ab.1</i>.</p

Skeletal defects in <i>unc45</i> mutants at 5 dpf.

<p>Ventral views of Alcian Blue stained cartilages (a–d). Wild type siblings (a), <i>unc45a^−/−</i> (b), <i>unc45b^−/−</i> (c), and <i>unc45b^−/−</i>; <i>unc45a^−/−</i> (d) mutants. Wild type siblings and <i>unc45a^−/−</i> mutants (a,b) have robust cartilage staining whereas <i>unc45b^−/−</i> (c) and <i>unc45b^−/−</i>; <i>unc45a^−/−</i> (d) mutants exhibit decreased staining, improper angling of the ceratohyal cartilages, and shortening of the palatoquadrates and Meckel's cartilage. The pectoral girdle (arrows) is missing or reduced in <i>unc45b^−/−</i> (c) and <i>unc45b^−/−</i>; <i>unc45a^−/−</i> (d) embryos. cb, ceratobranchial; ch, ceratohyal; hs, hyosymplectic; mc, Meckel's cartilage; pq, palatoquadrate. Small blue dots are an artifact of the fixation and staining process.</p

Comparison of <i>unc45a</i> and <i>unc45b</i> mRNA expression at 48 hpf.

<p>Wild type siblings (a,e,e'); <i>unc45a^−/−</i> (b,f,f'); <i>unc45b^−/−</i> (c,g,g'); and <i>unc45b^−/−</i>; <i>unc45a^−/−</i> (d,h,h') mutants. <i>unc45a</i> expression is diffuse in the region of the brain and pharyngeal arches. <i>unc45b</i> mRNA expression from lateral (e–h) and dorsal (e'–h') views, head to the left. Wild type siblings (e,e') and <i>unc45a^−/−</i> (f,f') mutants express <i>unc45b</i> in the extraocular (Eo), cardiac (Ca), trunk (Tr), and pectoral fin (Pf) muscles. Expression is either minimal or absent in the extraocular, cardiac, and pectoral fin muscles of the <i>unc45b^−/−</i> (g,g') and <i>unc45b^−/−</i>; <i>unc45a^−/−</i> (h,h') mutants while expression in the trunk musculature appears to up-regulated in these mutants. Apostrophes denote alternative views of the same embryo.</p

Phylogenetic analysis of Unc45 sequences.

<p>Protein sequences were aligned using ClustalW2 and phylogenetic trees were generated by the Neighbour-Joining method with MEGA5. Bootstrap values for 1000 replicates are indicated to the right of the nodes. Ce, <i>Caenorhabditis elegans</i>; Dm, <i>Drosophila melanogaster</i>; Dr, <i>Danio rerio</i>; Ga, <i>Gasterosteus aculeatus</i>; Gg, <i>Gallus gallus</i>; Hs, <i>Homo sapiens</i>; Mm, <i>Mus musculus</i>; Ol, <i>Oryzias latipes</i>; Tn, <i>Tetradon nigroviridis</i>; Tr, <i>Takifugu rubripes</i>; Xt, <i>Xenopus tropicalis</i>. The Ensembl gene IDs of the genes used in generating the phylogenetic trees are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048861#pone.0048861.s003" target="_blank">Table S1</a>.</p

Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility

<div><p>Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of <i>GIM3</i> did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.</p></div

Guk1-7 is thermally unstable.

<p>(A) Ribbon structure of Guk1 (PDB 1EX7). Positions of the four missense mutations and predicted ∆∆G values are indicated. Loss of fluorescence measured by flow cytometry after a two hour incubation at 37°C with cycloheximide is indicated in brackets. (B) Cellular thermal shift assay of Guk1 and Guk1-7 fused to a six histidine tag in lysates derived from cells grown at 25°C. One representative anti-His Western Blot is shown. The graph represents the means and standard deviations of Guk1 levels from three independent experiments. (C) Guk1 and Guk1-7 fused to a six histidine tag were expressed in cells grown at 25°C or shifted to 37°C for 20 min. Total cell lysate (T), soluble (S), and pellet fractions (P) were immunoblotted with anti-His antibodies.</p