Overexpression of Hsp104 by Causing Dissolution of the Prion Seeds Cures the Yeast [PSI+] Prion.

The yeast Sup35 protein misfolds into the infectious [PSI+] prion, which is then propagated by the severing activity of the molecular chaperone, Hsp104. Unlike other yeast prions, this prion is unique in that it is efficiently cured by the overexpression as well as the inactivation of Hsp104. However, it is controversial whether curing by overexpression is due to the dissolution of the prion seeds by the trimming activity of Hsp104 or the asymmetric segregation of the prion seeds between mother and daughter cells which requires cell division. To answer this question, we conducted experiments and found no difference in the extent of curing between mother and daughter cells when half of the cells were cured by Hsp104 overexpression in one generation. Furthermore, curing was not affected by the lack of Sir2 expression, which was reported to be required for asymmetric segregation of the [PSI+] seeds. More importantly, when either hydroxyurea or ethanol were used to inhibit cell division, the extent of curing by Hsp104 overexpression was not significantly reduced. Therefore, the curing of [PSI+] by Hsp104 overexpression is not due to asymmetric segregation of the prion seeds, but rather their dissolution by Hsp104.

Mechanisms for Curing Yeast Prions.

Prions are infectious proteins that self-propagate by changing from their normal folded conformation to a misfolded conformation. The misfolded conformation, which is typically rich in ß-sheet, serves as a template to convert the prion protein into its misfolded conformation. In yeast, the misfolded prion proteins are assembled into amyloid fibers or seeds, which are constantly severed and transmitted to daughter cells. To cure prions in yeast, it is necessary to eliminate all the prion seeds. Multiple mechanisms of curing have been found including inhibiting severing of the prion seeds, gradual dissolution of the prion seeds, asymmetric segregation of the prion seeds between mother and daughter cells during cell division, and degradation of the prion seeds. These mechanisms, achieved by using different protein quality control machinery, are not mutually exclusive; depending on conditions, multiple mechanisms may work simultaneously to achieve curing. This review discusses the various methods that have been used to differentiate between these mechanisms of curing.

Real-time imaging of yeast cells reveals several distinct mechanisms of curing of the [URE3] prion.

The [URE3] yeast prion is the self-propagating amyloid form of the Ure2 protein. [URE3] is cured by overexpression of several yeast proteins, including Ydj1, Btn2, Cur1, Hsp42, and human DnaJB6. To better understand [URE3] curing, we used real-time imaging with a yeast strain expressing a GFP-labeled full-length Ure2 construct to monitor the curing of [URE3] over time. [URE3] yeast cells exhibited numerous fluorescent foci, and expression of the GFP-labeled Ure2 affected neither mitotic stability of [URE3] nor the rate of [URE3] curing by the curing proteins. Using guanidine to cure [URE3] via Hsp104 inactivation, we found that the fluorescent foci are progressively lost as the cells divide until they are cured; the fraction of cells that retained the foci was equivalent to the [URE3] cell fraction measured by a plating assay, indicating that the foci were the prion seeds. During the curing of [URE3] by Btn2, Cur1, Hsp42, or Ydj1 overexpression, the foci formed aggregates, many of which were 0.5 µm or greater in size, and [URE3] was cured by asymmetric segregation of the aggregated seeds. In contrast, DnaJB6 overexpression first caused a loss of detectable foci in cells that were still [URE3] before there was complete dissolution of the seeds, and the cells were cured. We conclude that GFP labeling of full-length Ure2 enables differentiation among the different [URE3]-curing mechanisms, including inhibition of severing followed by seed dilution, seed clumping followed by asymmetric segregation between mother and daughter cells, and seed dissolution.

Heat shock protein 104 (Hsp104)-mediated curing of [PSI+] yeast prions depends on both [PSI+] conformation and the properties of the Hsp104 homologs.

Prions arise from proteins that have two possible conformations: properly folded and non-infectious or misfolded and infectious. The [PSI+] yeast prion, which is the misfolded and self-propagating form of the translation termination factor eRF3 (Sup35), can be cured of its infectious conformation by overexpression of Hsp104, which helps dissolve the prion seeds. This dissolution depends on the trimming activity of Hsp104, which reduces the size of the prion seeds without increasing their number. To further understand the relationship between trimming and curing, trimming was followed by measuring the loss of GFP-labeled Sup35 foci from both strong and weak [PSI+] variants; the former variant has more seeds and less soluble Sup35 than the latter. Overexpression of Saccharomyces cerevisiae Hsp104 (Sc-Hsp104) trimmed the weak [PSI+] variants much faster than the strong variants and cured the weak variants an order of magnitude faster than the strong variants. Overexpression of the fungal Hsp104 homologs from Schizosaccharomyces pombe (Sp-Hsp104) or Candida albicans (Ca-Hsp104) also trimmed and cured the weak variants, but interestingly, it neither trimmed nor cured the strong variants. These results show that, because Sc-Hsp104 has greater trimming activity than either Ca-Hsp104 or Sp-Hsp104, it cures both the weak and strong variants, whereas Ca-Hsp104 and Sp-Hsp104 only cure the weak variants. Therefore, curing by Hsp104 overexpression depends on both the trimming ability of the fungal Hsp104 homolog and the strength of the [PSI+] variant: the greater the trimming activity of the Hsp104 homolog and the weaker the variant, the greater the curing.

The multivesicular body is the major internal site of prion conversion.

The conversion of the properly folded prion protein, PrPc, to its misfolded amyloid form, PrPsc, occurs as the two proteins traffic along the endocytic pathway and PrPc is exposed to PrPsc. To determine the specific site of prion conversion, we knocked down various proteins in the endocytic pathway including Rab7a, Tsg101 and Hrs (also known as HGS). PrPsc was markedly reduced in two chronically infected cell lines by preventing the maturation of the multivesicular body, a process that begins in the early endosome and ends with the sorting of cargo to the lysosome. By contrast, knocking down proteins in the retromer complex, which diverts cargo away from the multivesicular body caused an increase in PrPsc levels. These results suggest that the multivesicular body is the major site for intracellular conversion of PrPc to PrPsc.

The clathrin-binding and J-domains of GAK support the uncoating and chaperoning of clathrin by Hsc70 in the brain.

Cyclin-G-associated kinase (GAK), the ubiquitously expressed J-domain protein, is essential for the chaperoning and uncoating of clathrin that is mediated by Hsc70 (also known as HSPA8). Adjacent to the C-terminal J-domain that binds to Hsc70, GAK has a clathrin-binding domain that is linked to an N-terminal kinase domain through a PTEN-like domain. Knocking out GAK in fibroblasts caused inhibition of clathrin-dependent trafficking, which was rescued by expressing a 62-kDa fragment of GAK, comprising just the clathrin-binding and J-domains. Expressing this fragment as a transgene in mice rescued the lethality and the histological defects caused by knocking out GAK in the liver or in the brain. Furthermore, when both GAK and auxilin (also known as DNAJC6), the neuronal-specific homolog of GAK, were knocked out in the brain, mice expressing the 62-kDa GAK fragment were viable, lived a normal life-span and had no major behavior abnormalities. However, these mice were about half the size of wild-type mice. Therefore, the PTEN-like domains of GAK and auxilin are not essential for Hsc70-dependent chaperoning and uncoating of clathrin, but depending on the tissue, these domains appear to increase the efficiency of these co-chaperones.

Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein-protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G-associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy-lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms.

Disruption of clathrin-mediated trafficking causes centrosome overduplication and senescence.

The Hsc70 cochaperone, G cyclin-associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin-dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin-mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin-dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication.

Hsp104 overexpression cures Saccharomyces cerevisiae [PSI+] by causing dissolution of the prion seeds.

The [PSI(+)] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI(+)], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI(+)]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI(+)] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI(+)] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.

Clathrin-coated vesicles from brain have small payloads: a cryo-electron tomographic study.

Clathrin coats, which stabilize membrane curvature during endocytosis and vesicular trafficking, form highly polymorphic fullerene lattices. We used cryo-electron tomography to visualize coated particles in isolates from bovine brain. The particles range from ∼66 to ∼134nm in diameter, and only 20% of them (all ⩾80nm) contain vesicles. The remaining 80% are clathrin "baskets", presumably artifactual assembly products. Polyhedral models were built for 54 distinct coat geometries. In true coated vesicles (CVs), most vesicles are offset to one side, leaving a crescent of interstitial space between the coat and the membrane for adaptor proteins and other components. The latter densities are fewer on the membrane-proximal side, which may represent the last part of the vesicle to bud off. A small number of densities - presumably cargo proteins - are associated with the interior surface of the vesicles. The clathrin coat, adaptor proteins, and vesicle membrane contribute almost all of the mass of a CV, with most cargoes accounting for only a few percent. The assembly of a CV therefore represents a massive biosynthetic effort to internalize a relatively diminutive payload. Such a high investment may be needed to overcome the resistance of membranes to high curvature.

Sequestration of Sup35 by aggregates of huntingtin fragments causes toxicity of [PSI+] yeast.

Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN(+)] prion, which is the amyloid form of Rnq1, or [PSI(+)] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI(+)] yeast than in [PIN(+)], even though HttQP103 formed multiple aggregates in both [PSI(+)] and [PIN(+)] yeast. This toxicity was only observed in the strong [PSI(+)] variant, not the weak [PSI(+)] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI(+)] prion is primarily due to sequestration of the essential protein, Sup35.

Endocytosis and clathrin-uncoating defects at synapses of auxilin knockout mice.

Neuronally expressed auxilin and ubiquitously expressed cyclin-G-dependent kinase (GAK) are homologous proteins that act as cochaperones to support the Hsc70-dependent clathrin uncoating of clathrin-coated vesicles. GAK was previously shown to be essential in mouse during embryonic development and in the adult. We have now engineered an auxilin knockout mouse. Mutant mice had a high rate of early postnatal mortality and surviving pups generally had a lower body weight than wild-type pups, although they had a normal life span. GAK was up-regulated as much as 3-fold in the brains of both surviving neonates and adult mutant mice. An increased number of clathrin-coated vesicles and empty cages were present at knockout synapses both in situ and in primary neuronal cultures. Additionally, clathrin-mediated endocytosis of synaptic vesicles in knockout hippocampal neurons was impaired, most likely due to sequestration of coat components in assembled coats and cages. Collectively, our results demonstrate the specialized role of auxilin in the recycling of synaptic vesicles at synapses, but also show that its function can be partially compensated for by up-regulation of GAK.

Hsp70 Binding to the N-terminal Domain of Hsp104 Regulates [PSI+] Curing by Hsp104 Overexpression.

Hsp104 propagates the yeast prion [PSI+], the infectious form of Sup35, by severing the prion seeds, but when Hsp104 is overexpressed, it cures [PSI+] in a process that is not yet understood but may be caused by trimming, which removes monomers from the ends of the amyloid fibers. This curing was shown to depend on both the N-terminal domain of Hsp104 and the expression level of various members of the Hsp70 family, which raises the question as to whether these effects of Hsp70 are due to it binding to the Hsp70 binding site that was identified in the N-terminal domain of Hsp104, a site not involved in prion propagation. Investigating this question, we now find, first, that mutating this site prevents both the curing of [PSI+] by Hsp104 overexpression and the trimming activity of Hsp104. Second, we find that depending on the specific member of the Hsp70 family binding to the N-terminal domain of Hsp104, both trimming and the curing caused by Hsp104 overexpression are either increased or decreased in parallel. Therefore, the binding of Hsp70 to the N-terminal domain of Hsp104 regulates both the rate of [PSI+] trimming by Hsp104 and the rate of [PSI+] curing by Hsp104 overexpression.

Mutations in Parkinsonism-linked endocytic proteins synaptojanin1 and auxilin have synergistic effects on dopaminergic axonal pathology.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by defective dopaminergic (DAergic) input to the striatum. Mutations in two genes encoding synaptically enriched clathrin-uncoating factors, synaptojanin 1 (SJ1) and auxilin, have been implicated in atypical Parkinsonism. SJ1 knock-in (SJ1-KIRQ) mice carrying a disease-linked mutation display neurological manifestations reminiscent of Parkinsonism. Here we report that auxilin knockout (Aux-KO) mice display dystrophic changes of a subset of nigrostriatal DAergic terminals similar to those of SJ1-KIRQ mice. Furthermore, Aux-KO/SJ1-KIRQ double mutant mice have shorter lifespan and more severe synaptic defects than single mutant mice. These include increase in dystrophic striatal nerve terminals positive for DAergic markers and for the PD risk protein SV2C, as well as adaptive changes in striatal interneurons. The synergistic effect of the two mutations demonstrates a special lability of DAergic neurons to defects in clathrin uncoating, with implications for PD pathogenesis in at least some forms of this condition.

The Properties and Domain Requirements for Phase Separation of the Sup35 Prion Protein In Vivo.

The Sup35 prion protein of budding yeast has been reported to undergo phase separation to form liquid droplets both at low pH in vitro and when energy depletion decreases the intracellular pH in vivo. It also has been shown using purified proteins that this phase separation is driven by the prion domain of Sup35 and does not re-quire its C-terminal domain. In contrast, we now find that a Sup35 fragment consisting of only the N-terminal prion domain and the M-domain does not phase separate in vivo; this phase separation of Sup35 requires the C-terminal domain, which binds Sup45 to form the translation termination complex. The phase-separated Sup35 not only colocalizes with Sup45 but also with Pub1, a stress granule marker protein. In addition, like stress granules, phase separation of Sup35 appears to require mRNA since cycloheximide treatment, which inhibits mRNA release from ribosomes, prevents phase separation of Sup35. Finally, unlike Sup35 in vitro, Sup35 condensates do not disassemble in vivo when the intracellular pH is increased. These results suggest that, in energy-depleted cells, Sup35 forms supramolecular assemblies that differ from the Sup35 liquid droplets that form in vitro.

Dopamine transporter and synaptic vesicle sorting defects underlie auxilin-associated Parkinson's disease.

Auxilin participates in the uncoating of clathrin-coated vesicles (CCVs), thereby facilitating synaptic vesicle (SV) regeneration at presynaptic sites. Auxilin (DNAJC6/PARK19) loss-of-function mutations cause early-onset Parkinson's disease (PD). Here, we utilized auxilin knockout (KO) mice to elucidate the mechanisms through which auxilin deficiency and clathrin-uncoating deficits lead to PD. Auxilin KO mice display cardinal features of PD, including progressive motor deficits, α-synuclein pathology, nigral dopaminergic loss, and neuroinflammation. Significantly, treatment with L-DOPA ameliorated motor deficits. Unbiased proteomic and neurochemical analyses of auxilin KO brains indicated dopamine dyshomeostasis. We validated these findings by demonstrating slower dopamine reuptake kinetics in vivo, an effect associated with dopamine transporter misrouting into axonal membrane deformities in the dorsal striatum. Defective SV protein sorting and elevated synaptic autophagy also contribute to ineffective dopamine sequestration and compartmentalization, ultimately leading to neurodegeneration. This study provides insights into how presynaptic endocytosis deficits lead to dopaminergic vulnerability and pathogenesis of PD.

Clathrin-independent internalization of normal cellular prion protein in neuroblastoma cells is associated with the Arf6 pathway.

To understand the role of clathrin-mediated endocytosis in the internalization of normal cellular prion protein (PrP(c)) in neuronal cells, N2a cells were depleted of clathrin by RNA interference. PrP(c) internalization via the constitutive endocytic pathway in the absence of Cu(2+) and the stimulated pathway in the presence of Cu(2+) were measured in both control and clathrin-depleted cells. Depletion of clathrin had almost no effect on the internalization of PrP(c) either in the presence or absence of Cu(2+), in contrast to the marked reduction observed in transferrin uptake. By contrast, the internalization of PrP(c) was inhibited by the raft-disrupting drugs filipin and nystatin, and by the dominant-negative dynamin-1 mutant dynamin-1 K44A, both in the presence and absence of Cu(2+). The internalized PrP(c) was found to colocalize with cargo that traffic in the Arf6 pathway and in large vacuoles in cells expressing the Arf6 dominant-active mutant. These results show that PrP(c) is internalized in a clathrin-independent pathway that is associated with Arf6.

Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast.

Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis.

OBJECTIVE: To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor-differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). METHODS AND RESULTS: We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. CONCLUSIONS: Our findings show that human macrophage colony-stimulating factor-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

Huntingtin Polyglutamine Fragments Are a Substrate for Hsp104 in Saccharomyces cerevisiae.

The aggregation of huntingtin fragments with expanded polyglutamine repeat regions (HttpolyQ) that cause Huntington's disease depends on the presence of a prion with an amyloid conformation in yeast. As a result of this relationship, HttpolyQ aggregation indirectly depends on Hsp104 due to its essential role in prion propagation. We find that HttQ103 aggregation is directly affected by Hsp104 with and without the presence of [RNQ+] and [PSI+] prions. When we inactivate Hsp104 in the presence of prion, yeast cells have only one or a few large HttQ103 aggregates rather than numerous smaller aggregates. When we inactivate Hsp104 in the absence of prion, there is no significant aggregation of HttQ103, whereas with active Hsp104, HttQ103 aggregates accumulate slowly due to the severing of spontaneously nucleated aggregates by Hsp104. We do not observe either effect with HttQ103P, which has a polyproline-rich region downstream of the polyglutamine region, because HttQ103P does not spontaneously nucleate and Hsp104 does not efficiently sever the prion-nucleated HttQ103P aggregates. Therefore, the only role of Hsp104 in HttQ103P aggregation is to propagate yeast prion. In conclusion, because Hsp104 efficiently severs the HttQ103 aggregates but not HttQ103P aggregates, it has a marked effect on the aggregation of HttQ103 but not HttQ103P.