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FASEB J. Feb 2009; 23(2): 451–463.
PMCID: PMC2630789

Abnormal proteins can form aggresome in yeast: aggresome-targeting signals and components of the machinery

Abstract

In mammalian cells, abnormal proteins that escape proteasome-dependent degradation form small aggregates that can be transported into a centrosome-associated structure, called an aggresome. Here we demonstrate that in yeast a single aggregate formed by the huntingtin exon 1 with an expanded polyglutamine domain (103QP) represents a bona fide aggresome that colocalizes with the spindle pole body (the yeast centrosome) in a microtubule-dependent fashion. Since a polypeptide lacking the proline-rich region (P-region) of huntingtin (103Q) cannot form aggresomes, this domain serves as an aggresome-targeting signal. Coexpression of 103Q with 25QP, a soluble polypeptide that also carries the P-region, led to the recruitment of 103Q to the aggresome via formation of hetero-oligomers, indicating the aggresome targeting in trans. To identify additional factors involved in aggresome formation and targeting, we purified 103QP aggresomes and 103Q aggregates and identified the associated proteins using mass spectrometry. Among the aggresome-associated proteins we identified, Cdc48 (VCP/p97) and its cofactors, Ufd1 and Nlp4, were shown genetically to be essential for aggresome formation. The 14-3-3 protein, Bmh1, was also found to be critical for aggresome targeting. Its interaction with the huntingtin fragment and its role in aggresome formation required the huntingtin N-terminal N17 domain, adjacent to the polyQ domain. Accordingly, the huntingtin N17 domain, along with the P-region, plays a role in aggresome targeting. We also present direct genetic evidence for the protective role of aggresomes by demonstrating genetically that aggresome targeting of polyglutamine polypeptides relieves their toxicity.—Wang, Y., Meriin, A. B., Zaarur, N., Romanova, N. V., Chernoff, Y. O., Costello, C. E., Sherman, M. Y. Abnormal proteins can form aggresome in yeast: aggresome-targeting signals and components of the machinery.

Keywords: polyglutamine, aggregation, VCP, 14-3-3 protein, neurodegeneration, misfolding

Special mechanisms of refolding and selective degradation have evolved to protect cells from the accumulation of mutant and damaged polypeptides. If these cellular mechanisms fail, abnormal proteins aggregate. Investigations into the mechanisms of intracellular protein aggregation are attracting growing attention in biology and medicine because of their relevance to a number of neuropathological conditions. In many major neurodegenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, the pathology and the eventual death of specific neuronal populations occur because of the accumulation of certain abnormal polypeptides, which can aggregate and form insoluble intracellular inclusion bodies (IBs) (for review see ref. 1). The formation of the IBs generally precedes neurodegeneration and cell death. Such observations initially led to the widely held assumption that polypeptide aggregation is the critical event that triggers neuropathology, at least in some of these diseases. On the other hand, there have been several reports that small aggregates or oligomers of abnormal proteins cause toxicity, whereas the formation of IBs actually protects cells (2). These contradictions could be in part due to the fact that IBs may represent a heterogenous group and include aggregates of different types and with different effects.

It was initially assumed that protein aggregation is a spontaneous process, resulting from a natural tendency of unfolded polypeptides to associate with each other. However, it has recently become clear that there are multiple aggregation pathways, some involving cellular factors. In certain aggregation pathways, small aggregates of misfolded polypeptides in the cytoplasm of mammalian cells may converge, via microtubule-based transport, at the centrosome, forming a large depot of protein aggregates (3,4,5,6), called an aggresome (6, 7). It was demonstrated that chaperones and components of the ubiquitin-proteasome system (UPS) are recruited to these aggresomes (7,8,9), which suggests that aggregated polypeptides could be refolded or degraded via these pathways. Furthermore, autophagy was found to facilitate degradation of the aggresome-associated misfolded proteins (10,11,12). Recently a number of factors have been reported to participate in aggresome formation, including the microtubule-associated histone deacetylase HDAC6 (13), ubiquitin binding proteins PLIC, ataxin 3 and p62/sequestosome (14,15,16,17), and the ubiquitin ligase Parkin (18).

It has been suggested that aggresome formation represents a protective response of cells to a buildup of aggregating abnormal polypeptides under conditions in which chaperones and UPS machineries fail to adequately dispose the abnormal species (1, 19). In line with this concept, the appearance of an aggresome usually occurs in mammalian cells subsequent to inhibition of the proteasome (7). The idea that aggresomes are cytoprotective is supported generally by indirect evidence, based on the correlation between aggresome formation and cell survival and on the enhancement of the toxicity of various abnormal proteins by inhibitors of microtubule-dependent transport (8, 20). Therefore, to validate this hypothesis, direct testing of the protective function of aggresomes is critical. It should be noted that, although protein inclusions in various diseases, such as Lewy bodies or Mallory bodies, have been suggested to represent aggresomes (21,22,23,24), in vivo evidence that these inclusion bodies are truly aggresomes has not been presented, and it therefore remains possible that these inclusions may result from other aggregation pathways.

Many reports concerning aggregation of pathological and other abnormal proteins do not make a clear distinction between aggresome formation and other aggregation pathways. To understand these processes, it is necessary to define the aggresome pathway using mechanism-based criteria. Considering that aggresome formation involves a microtubule-dependent transport of small aggregates to the centrosome, as originally demonstrated by Kopito and colleagues (7), the criteria for an aggresome should include the microtubule dependence and localization at a centrosomal site. On the other hand, some other features of aggresomes are common to various types of aggregates, such as their association with chaperones or components of the UPS. These factors are not reported to be critical for aggresome formation and should thus not be used as defining criteria for aggresomes.

In our studies of aggregation of abnormal proteins and their effects on cells, we have focused on polypeptides with expanded polyglutamine domains (polyQ). We have developed a yeast model that reproduces both polyQ length-dependent aggregation and cytotoxicity and thus allows the genetic investigation of cellular components that are involved in protein aggregation (25), a goal much less attainable with mammalian models. In this model we initially expressed green fluorescent protein (GFP) -tagged constructs carrying exon1 of the huntingtin gene lacking the proline-rich domain (P-region) under the control of the GAL1 promoter (see Fig. 1A for the constructs used in this work). This polypeptide construct, with a polyQ domain of normal size (25Q), does not form detectable aggregates and is not toxic to yeast cells. In contrast, a similar polypeptide, but with an expanded polyQ (103Q), forms multiple aggregates in every cell and is toxic. On the other hand, a polypeptide derived from the complete huntingtin exon 1 containing the P-region and an expanded polyQ (103QP) forms a single large aggregate in yeast cells and is nontoxic (26,27,28). We and others have demonstrated that the initial steps in aggregation of 103Q or other polyQ-expanded constructs, and the ability of these constructs to form multiple small aggregates in the cytosol, require the presence of a prion form of some of the yeast proteins with QN-rich domains, such as Rnq1, New1, or Sup35 (25, 29,30,31,32). Furthermore, we have found that, in addition to prions, the initial stages of polyQ aggregation require components of the endocytosis machinery (26). However, our previous studies did not address processes related to formation of an aggresome from small aggregates.

Figure 1.
103QP forms aggresome in yeast. A) Scheme of major constructs used in this study. B) Benomyl treatment (25 μg/ml added simultaneously with induction of 103QP) disrupts formation of a single aggregate by 103QP. Top panel: fluorescent microscopy ...

Here we demonstrated that the single aggregate formed by 103QP in yeast represents a bone fide aggresome and that its P-region serves as an aggresome-targeting signal. Our data also show that aggresome formation is cytoprotective. Through a combination of biochemical and genetic studies, we identified several novel players in aggresome formation. By establishing the yeast aggresome as a model system, this work offers a tool for rapid dissection of mechanisms of aggresome formation by genetic methods.

MATERIALS AND METHODS

Strains and constructs

Strains with GFP-tagged endogenous proteins and HIS3 marker in a parental strain MATα his3Δ leu2Δ met15Δ ura3Δ were obtained from the Yeast GFP Clone Collection (Invitrogen, Carlsbad, CA, USA). Deletion mutants of the wild-type strains BY4739 (MATα leu2Δ lys2Δ ura3Δ) or BY4742 (MATá his3Δ leu2Δ lys2Δ ura3Δ) were obtained from the deletion library (Invitrogen) of yeast nonessential genes (33). Other mutant and the corresponding wild-type strains are listed in Table 1.

TABLE 1.
Additional strains used in this study

Strain GT1118-2B was isolated as a G418R His+ spore clone in the meiotic progeny obtained after mating the bmh1Δ strain from the Invitrogen deletion library to the isogenic strain of the opposite mating type S288C Cdc48-GFP, followed by sporulation and dissection.

Constructs for expression of polyQ polypeptides under the control of the Gal1 promoter were described previously (26) Briefly, either complete huntingtin exon1 or exon 1 lacking the proline-rich domain were FLAG-tagged at the N terminus and tagged with enhanced GFP (EGFP) at the C terminus (Fig. 1A) [for certain applications, EGFP was substituted with monomeric red fluorescent protein (mRFP) or cyan fluorescent protein (CFP)]. Vectors pYES2 and pYES3 (Invitrogen) were routinely used. For Cu2+-regulated expression, we used the pCUP1 vector.

Constructs for expression of polyQ polypeptides under the control of the CMV promoter were described previously (34). Human embryonic kidney (HEK) 293 cells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS heat inactivated. Cells were transfected with 0.02 μg of plasmid encoding 103Q or 103QP using Lipofectamine 2000 reagent (Invitrogen) according to the manufacture’s protocol.

Yeast growth and induction

Cells were routinely grown at 30°C on selective minimal medium with 2% raffinose and, for induction of 103Q, were transferred into the selective media with 2% galactose (SG) for 4–6 h. For CUP promoter induction, 100 μM Cu2+ was added for 4 h. For the heat-sensitive mutants, cdc48-10, npl4-1, ufd1, and uba1-204 were grown at 37°C after induction of PolyQ. For the cold-sensitive, cdc48-1 was grown at 25°C after induction of PolyQ.

Antibodies

The following primary antibodies were used in this work: anti-FLAG monoclonal antibody (Sigma-Aldrich Corp., St Louis, MO, USA); anti-GFP (B-34) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-GFP antibody (Clontech Laboratories Inc., Palo Alto, CA, USA); rabbit anti-Bmh1/Bmh2 polyclonal antibody (gift from Dr. Sandra Lemmon, University of Miami, Miami, FL, USA); rabbit anti-cdc48 antibody (gift from Dr. Alexander Buchberger, Max Planck Institute of Biochemistry, Martinsried, Germany). Secondary antibodies used for immunoblotting were horseradish peroxidase (HRP) -linked anti-rabbit or anti-mouse immunoglobulin Gs (IgGs; GE Healthcare, Piscataway, NJ, USA) or rabbit true blot HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology); for immunoprecipitation of polyQ aggregates, AffiniPure Rabbit anti-mouse and goat anti-rabbit IgGs (H+L) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA).

Immunoprecipitation and immunoblotting

Yeast cells from 200 ml culture were disrupted at 4°C by 10-min vortexing with 425–600 μm acid-washed glass beads in lysis buffer (50 mM Tris HCl, pH 7.4; 150 mM NaCl; 1% Triton X-100; 1 mM EDTA; 5 mM PMSF; 1 mM benzamidine; 5 μg/mL of each: leupeptin, pepstatin A, aprotinin). After centrifugation at 10,000 g, the supernatants were incubated with either anti-FLAG or anti-GFP polyclonal antibody at 4°C for 1 h, and the complexes then were pulled down with protein G-Sepharose beads (Santa Cruz). The bound proteins were analyzed by SDS-PAGE followed with immunoblotting.

Fluorescence microscopy

Fluorescence microscopy was performed with an Axiovert 200 (Carl Zeiss, Oberkochen, Germany) microscope with an ×100 objective using the manufacturer’s software. For the colocalization study of Cdc48 with PolyQs, the cells were fixed for 10 min in 4% formaldehyde, washed with PBS, and analyzed on the glass slides.

For aggregation quantification, the transfected cells were counted by a masked observer and categorized to diffused, one inclusion, and multiple aggregates. The number of cells containing any inclusions was expressed as a percentage of total transfected cells. Each experiment was repeated 3 times, and each time 200 cells were counted.

Isolation of aggregates

The isolation procedure was described previously (35). Briefly, yeast cells expressing 103Q or 103QP grown to the logarithmic phase were disrupted with glass beads in lysis buffer (50 mM Hepes, pH 7.5; 150 mM NaCl; 1% TritonX-100 with protease inhibitors). Heavy particles were separated by size-exclusion chromatography on Sephacryl S-400 HR (GE Healthcare; 50 ml column, flow rate 0.85 ml/min). Usually 4 fractions with the highest content of aggregates (tested by fluorescence microscopy) were combined. The corresponding fractions from the cell lysates with vector only were used as a control. Mouse anti-FLAG IgG, rabbit anti-mouse IgG, and goat anti-rabbit IgG were added consecutively, as described in ref. 35. Finally, the samples were loaded onto the 50% sucrose cushion and spun down at 600 g for 2.5 min. The supernatant was aspirated, and the purified aggregate pellet was stored at −20°C.

Two-dimensional polyacrylamide (2-D) gel electrophoresis

Purified aggregate complexes (300–500 μg) were resuspended in 200 μl rehydration buffer (Bio-Rad Laboratories, Hercules, CA, USA) and separated with 2-D gel electrophoresis. Isoelectric focusing (IEF) was performed by using immobilized pH gradient strips with a pH range from 3 to 10 on a Bio-Rad IEF cell with a programmed voltage gradient. SDS-PAGE was performed on Bio-Rad precast 12.5% polyacrylamide gel. Gels were stained overnight with Coomassie blue.

Mass spectrometry and protein identification

Gel spots of interest were excised, destained, dithiothreitol-reduced, modified with iodoacetamide, and digested with trypsin. After the peptides were extracted from gel pieces using 1% trifluroacetic acid (TFA) and 50% acetonitrile, the solutions were ZipTip™ cleaned (Millipore, Billerica, MA, USA) and taken to dryness. The peptides were resuspended with 0.1% TFA and 50% acetonitrile and spotted onto the matrix-assisted laser-desorption/ionization (MALDI) target with 2,5-dihydroxybenzoic acid as matrix. The resulting tryptic peptides of each spot were analyzed with a Bruker Reflex IV matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) (Bruker, Billerica, MA, USA) equipped with a nitrogen laser (Laser Science Inc., Franklin, MA, USA) having a 3-ns pulse width at 337 nm. Spectra were acquired by summing the signals recorded after 150–200 laser shots. Singly charged monoisotopic peptide masses were generated and used as inputs for database searching using MoverZ software (ProteoMetrics, New York, NY, USA), after external and internal calibration of spectra (36). Database searching was performed against the NCBInr database by using the online ProFound search engines (http://prowl.rockefeller.edu/prowl-cgi/profound.exe; accessed 2006) (37).

Search parameters were as follows: Saccharomyces cerevisiae for the taxonomic category; protein mass range of 0–200 kDa; iodoacetamide modified cysteines; maximum of 2 missed cleavage sites; mass tolerance of 0.1 Da.

Tandem MS/MS analyses were performed on an electrospray ionization (ESI) QSTAR Pulsar i quadrupole-orthogonal TOF MS (MS and MS/MS) (Applied Biosystems, Foster City, CA, USA). Peptides cleaned by ZipTip were eluted with acetonitrile:water:formic acid (50:50:0.5, v/v/v) and analyzed. Collision-induced decomposition MS/MS spectra were acquired at 30–60 V collision cell voltage, and the resulting spectra were examined manually. The data were analyzed using QAnalyst (Applied Biosystems) software.

His pulldown assay

The His pulldown experiments were conducted by using Ni-NTA resin (Novagen, Madison, WI, USA). Yeast culture (200 ml) of His6-Ubiquitin or His6-Smt3 proteins (100 μM) coexpressed with pYES3-103Q and pYES3-103QP were induced with 100 mM CuSO4 and 2% galactose for 4 h. Cells were lysed in lysis buffer (50 mM Tris HCl, pH 7.4; 150 mM NaCl; 1% Triton X-100; 1 mM EDTA; 5 mM PMSF; 1 mM benzamidine; 5 μg/mL of each: leupeptin, pepstatin A, aprotinin, 20mM N-ethylmaleimide). Lysates (300 μl) from 10,000 g centrifugation were incubated with 40 μl of Ni-NTA resin for 1 h at 4°C. After washing the Ni-NTA resins with phosphate sodium buffer plus 20 mM imidazole (pH 8.0) 6 times, bound proteins were eluted with 50 μl of 250 mM imidazole and analyzed by SDS-PAGE.

RESULTS

Aggresome formation in yeast

As described in the introduction, a dramatic difference in the aggregation patterns in yeast of polyQ polypeptides with or without the P-region was previously demonstrated: 103Q polypeptide forms multiple aggregates and is toxic, whereas 103QP forms a single large aggregate and is not toxic (26,27,28) (see also Fig. 1B).To investigate whether these phenotypic differences result from differences in the mechanisms of aggregation of 103QP and 103Q, we tested whether either of these processes requires microtubules. Accordingly, 103QP and 103Q were induced for 6 h in the presence or absence of benomyl, an inhibitor of microtubular polymerization. This treatment completely blocked formation of single large 103QP aggregates, and instead multiple small aggregates appeared in the yeast cells (Fig. 1B), indicating that emergence of the single aggregate requires microtubules. On the other hand, formation of multiple small aggregates by 103Q was not affected by nocodazole (not shown).

We further tested whether this aggregate colocalizes with the spindle pole body (SPB), the prototype of the centrosome in yeast (38). In this experiment we utilized clones expressing GFP-tagged components of the SPB, including Tub4 and Spc72. These clones were obtained from the collection of yeast strains with GFP-tagged endogenous protein expressed at normal levels. The colocalization studies used 103QP tagged with the red fluorescent protein mRFP (103QP-RFP), so this polypeptide could be distinguished from the GFP-tagged endogenous proteins. Of note, replacement of the fluorescent polypeptide to mRFP did not significantly change the aggregation pattern. In the majority of cells, a single aggregate was formed in a benomyl-sensitive manner, demonstrating that the new tag did not affect the dependency on microtubules of the 103QP aggregate (Supplemental Fig. 1). Concurrently, benomyl also destroyed SPB (labeled by γ-tubulin) (not shown), illustrating the effectiveness of treatment. In almost all cells that formed a single 103QP-RFP aggregate, it colocalized with Spc72 and γ-tubulin (Tub4) (Fig. 1C). Interestingly, in dividing cells with 2 SPBs, 103QP aggregates were often seen associated only with 1 SPB, a result in line with the asymmetrical distribution of the aggresome reported to occur during division of mammalian cells (39). In contrast, no colocalization with components of SPB was observed with multiple 103Q-RFP aggregates (not shown). Thus, since the single 103QP aggregate colocalized with the centrosome prototype and its formation required microtubules, we concluded that the 103QP aggregate represents an aggresome. It is noteworthy that in contrast to 103Q, 103QP does not cause cell toxicity in yeast (27), suggesting the protective function of aggresome formation.

An important conclusion from these data is that the P-region of the huntingtin exon 1 plays a critical role in aggresome targeting. On the other hand, aggresome targeting of the polypeptide with extended polyQ could occur even in the absence of the P-region, but only when 103Q is expressed at low levels. This targeting (Fig. 1D) could be detected only on expression of 103Q under the control of methionine-regulated promotor at suboptimal induction conditions (100 μM methionine for 4 h), resulting in expression of 103Q reaching no more than 1/20 of the levels seen with galactose-inducible promotor. Notably, under these conditions, formation of single aggregates was blocked by benomyl (Fig. 1D). It is noteworthy that with galactose induction, the levels of 103QP were even higher than those of 103Q (Fig. 1E). Moreover, under optimal induction conditions in the Met-regulated system, no aggresomes were seen (not shown). Thus, the P-region confers a dramatic increase in the efficiency of aggresome targeting.

P-region targets polyQ aggregates to an aggresome in trans

The data described above indicate that the P-region can target polyQ polypeptides to the aggresome, thereby suggesting the novel concept of special aggresome-targeting segments in aggregating polypeptides. This concept supports that an aggregating polypeptide incompetent in aggresome targeting still can be forced into the aggresome on interacting with a protein that carries an aggresome-targeting sequence.

In contrast to 103QP, 25QP is soluble and does not form any detectable aggregates. However, 25QP can interact with a polypeptide with expanded polyQ domains, as seen in mammalian cells (40). We hypothesized that if 103Q could recruit soluble 25QP into mixed oligomers/aggregates in yeast, the P-region of 25QP would be able to target such structures to the aggresome. To test this possibility, we investigated whether 25QP forms mixed oligomers with 103Q in yeast. Accordingly, we coexpressed 25QP (GFP tagged) and 103Q-RFP and immunoprecipitated 25QP with anti-GFP antibody from clarified cell lysates. The presence of 103Q-RFP in complexes with 25QP was assayed using an antibody against the FLAG-tag, which is present in both polypeptides. 103Q-RFP coprecipitated with 25QP (Fig. 2A), indicating formation of mixed 25QP/103Q oligomers.

Figure 2.
Proline-rich region targets polyQ aggregates to aggresome in trans. Fluorescent microscopy images of live cells. A) 103Q forms a complex with 25QP. Immunoblot with anti-FLAG antibody (detects both 103Q and 25QP). Cells expressing 103Q-RFP alone or together ...

Further, we tested whether 25QP in mixed oligomers can provide the aggresome targeting for 103Q. To have 25QP in cells prior to induction of 103Q, we expressed 25QP under the control of GAL1 promoter, while expression of 103Q-RFP was controlled by CUP1 promoter. Cells were grown on selective medium with galactose for 2 h to express 25QP-GFP, and then 103Q-RFP was induced for 6 h with 100 μM of Cu2+ in the same medium. Using fluorescent microscopy, we assessed whether coexpression with 25QP can force 103Q to form an aggresome. Indeed, coexpression with 25QP-GFP led to formation of single 103Q-RFP aggregates (Fig. 2B) in the majority of cells with aggregates (Fig. 2C). Notably, formation of the single aggregates was blocked by the microtubule-disrupting agent benomyl (Fig. 2B). In these aggregates, 103Q-RFP colocalized with 25QP (not shown). In contrast, coexpression with 25Q-GFP did not affect the aggregation pattern of 103Q-RFP, which still formed multiple aggregates (Fig. 2B). Therefore, in this system the extended polyQ domain of 103Q facilitates aggregation, while the P-region of 25QP promotes aggresome targeting in trans. Of note, targeting in trans by an interacting endogenous protein carrying an aggresome-targeting domain may be responsible for the limited aggresome formation of 103Q expressed at lower levels, described above.

The idea of 25QP-mediated targeting of 103Q to an aggresome is in agreement with recently published data that coexpression of 25QP suppresses toxicity of 103Q (27). In fact, coexpression with 25QP, but not with 25Q, completely relieved the 103Q toxicity (Supplemental Fig. 2) without reducing its levels (Fig. 2A, lanes 1 and 2). Coexpression of 103QP, similarly to 25QP, blocked the 103Q-induced toxicity (not shown). These data indicate that forcing a toxic polypeptide to an aggresome can suppress the toxicity.

Identification of aggresome-associated proteins

Our observation that 103QP forms an aggresome while 103Q forms multiple aggregates suggests a powerful approach toward identification of components of the aggresome machinery via analysis of proteins associated with these distinct types of aggregates. Accordingly, we employed our recently developed method for purification of protein aggregates under mild conditions, a strategy based on affinity isolation without a solid support (35). Briefly, high-molecular-weight fractions from cell lysates were isolated by size-exclusion chromatography. These fractions were incubated with an anti-GFP antibody that decorated aggregates. This incubation was followed by sequential incubations with secondary and tertiary antibodies that formed a heavy antibody mesh with impregnated aggregates. Finally, this mesh was separated from other structures by slow centrifugation through a sucrose cushion (see Materials and Methods). The resulting fraction contained aggregated polyglutamine polypeptides together with associated proteins and IgG (40). Aggregates were isolated from cells expressing either 103Q (small aggregates) or 103QP (aggresomes) (see Materials and Methods), and the aggregate-associated proteins were separated by 2-D SDS-PAGE (Fig. 3A). As a negative control, we used lysates of cells transformed with an empty vector.

Figure 3.
Identification of aggresome-associated proteins. A) Isolated 103QP aggresomes and 103Q multiple aggregates separated on 2-D gel (Coomassie blue staining). Spots of Cdc48 and Bmh1 are circled. B) Representative MALDI-TOF mass spectrum shows the peptide ...

Spots on the 2-D gel representing single aggregate-associated proteins were excised and identified by MALDI-TOF MS peptide mass mapping and nanoflow liquid chromatography ESI tandem MS on a quadrupole orthogonal TOF MS. Figure 3B provides an example of a protein identified unambiguously. The top panel shows the MALDI-TOF mass spectrum leading to the peptide fingerprinting of Bmh1. The tandem mass spectrum (bottom panel) of the peptide [M+2H]2+ m/z 746.4 observed in the nanospray mass spectrum (which corresponds to the singly-charged [M+H]+ at m/z 1491.7 in the MALDI mass spectrum shown in the top panel) confirmed that the peptide contains residues 29–42 from Bmh1. The N terminus of Bmh1 was found to be acetylated.

Table 2 shows the list of proteins found to be associated with 103Q and/or 103QP aggregates. Most of the identified proteins belong to the groups of either molecular chaperones or glycolytic enzymes. The members of the latter group associated with either type of the aggregates. The presence of these glycolytic enzymes in the aggregates probably results from their high sensitivity to oxidation and propensity for aggregation (41). This could explain their association with polyQ aggregates, and therefore they are unlikely to play an active role in aggregation of polyQ polypeptides. On the other hand, many of the glycolitic enzymes identified here were found to be associated with huntingtin in a polyQ-length-dependent manner in mammalian cells (42). Certain chaperones, including Sis1, Hsp42, and chaperone cofactor Sgt2, were found to be associated with both 103Q and 103QP aggregate isolates. In contrast, recruitment of other chaperones, as well as of several other proteins, differed between 103Q and 103QP aggregates. For example, spots corresponding to Hsp90 or Sse1 were reproducibly detected on the 2-D gels with isolated 103QP aggregates but not with 103Q aggregates (sample loading on these gels was normalized by the amount of corresponding polyQ), whereas Ssa1 or Ssb1 spots were found associated exclusively with 103Q aggregates. Interestingly, association of chaperones with polyQ polypeptides was previously found to be affected by the aggregation state (35). Identification of proteins specifically associated with the aggresome provided the basis for the following genetic tests that explored their roles in aggresome formation.

TABLE 2.
Comparison of proteins associated with 103Q and 103QP aggregates identified by mass spectrometry

Cdc48 (VCP/p97) and its cofactors play a critical role in aggresome formation

Factors involved in aggresome targeting may be present among the proteins specifically associated with 103QP aggresomes. Accordingly, in comprehensive genetic testing, we examined the effects of deletions of the corresponding genes on formation of 103QP aggresome. For these experiments we used strains from the collection of deletions of yeast nonessential genes (see Materials and Methods). Mutant strains were transformed with 103QP-expressing plasmid, and formation of 103QP aggresome was evaluated after 6 h induction. These experiments showed that aside from bmh1 (discussed below), other tested deletions did not or just mildly affected 103QP aggregation.

Among polypeptides specifically associated with aggresomes, we identified an essential protein Cdc48, the yeast homologue of mammalian VCP/p97, an AAA chaperone involved in ubiquitination of certain substrates (43). Notably, VCP/p97 was found to be associated with polyQ aggregates in mammalian cells (44). VCP/p97 also appears to play a role in formation of an aggresome by a bulk of ubiquitinated polypeptides in response to proteasome inhibitors (45).

The presence of Cdc48 in 103QP aggregates detected by mass spectrometry was confirmed by immunoblotting of the purified aggregates with anti-Cdc48 antibody (Fig. 4A). Furthermore, in a strain expressing Cdc48-GFP (from the collection of yeast clones with GFP-tagged endogenous proteins; see above) this protein clearly colocalized with the aggresome formed by 103QP-RFP, and no colocalization was seen with the multiple aggregates formed by 103Q-RFP (Fig. 4B). On the other hand, we did not detect significant association of Cdc48 (VCP/p97) with soluble 103QP, 103Q, or 25QP in immunoprecipitates from clarified lysates (Supplemental Fig. 3). This suggests that either such association is weak or unstable, or Cdc48 (VCP/p97) associates only with aggregates at later stages of aggresome formation.

Figure 4.
Cdc48 is critical for aggresome formation. A) Cdc48, but not Bmh1, associates specifically with 103QP aggregates. Immunoblot of purified 103QP and 103Q aggregates and control (labeled on the top) developed with anti-Cdc48, anti-Bmh1, or anti-GFP (for ...

Because Cdc48 is an essential protein, there was no corresponding strain in the deletion collection. Therefore, to test for the role of Cdc48 (VCP/p97) in aggresome formation genetically, heat-sensitive cdc48 mutant cdc48-10 and cold-sensitive mutant cdc48-1 were transformed with 103QP, and aggresome formation was examined after 6 h induction at the respective nonpermissive temperatures. Both mutants manifested significant defects in this process, and 103QP formed multiple small aggregates in almost 100% of cdc48-1 cells, and in more than 80% of cdc48-10 cells (compared to less than 10% in the isogenic wild-type strains under the respective conditions) (Fig. 4C and not shown). In cdc48-1 mutant, the aggresome formation by 103Q expressed at low levels from MET promoter was also suppressed (Supplemental Fig. 4). Therefore, Cdc48 chaperone appears to be critical for aggresome formation. We also observed significant 103QP, but not 25QP (not shown), toxicity in cdc48 mutants even at the permissive temperatures (Fig. 4D), indicating that even partial prevention of aggresome formation exposes the toxicity of an abnormal protein.

Cdc48 (VCP/p97) is involved in multiple cellular processes, including ubiquitination of certain short-lived proteins (46, 47). In this process, Cdc48 associates with many factors that control ubiquitination (46, 48). Multiple Cdc48-interacting proteins have been identified in yeast, using either high throughput 2-hybrid analysis or analysis of protein complexes (49, 50). To identify Cdc48 cofactors involved in the 103QP aggresome formation, we tested more than 30 mutants in the genes of known Cdc48-associated factors. Among these mutants, only temperature-sensitive mutations in 2 ubiquitination cofactors, Ufd1 and Nlp4, strongly suppressed emergence of the aggresome (Fig. 4E) and facilitated 103QP toxicity (not shown). Therefore it appears that Cdc48 (VCP/p97) cooperates with Ufd1 and Nlp4 in aggresome formation.

The role of Cdc48-Nlp4-Ufd1 in aggresome formation appears to be distinct from substrate ubiquitination

As mentioned above, Cdc48, Npl4, and Ufd1 cooperate in ubiquitination of certain substrates, suggesting that 103QP ubiquitination may be critical for aggresome targeting. This hypothesis would be consistent with a previously proposed model of aggresome targeting, where ubiquitin-binding protein HDAC6 binds to small aggregates of ubiquitinated polypeptides and recruits them to the dynein motor complex for further transport to an aggresome (13). Accordingly, we examined whether polyglutamine polypeptides undergo ubiquitination. N-terminally His-tagged ubiquitin was coexpressed with 103QP, 103Q, or 25QP. Cells were lysed in the presence of N-ethylmaleimide to block deubiquitinating enzymes, and ubiquitin conjugates were isolated using Ni2+-column, then immunoblotted with anti-GFP antibody to detect conjugates of polyQ polypeptides. Despite the massive presence of ubiquitin conjugates in the lysates, we could not detect anti-GFP antibody-reacting bands (Supplemental Fig. 5A). The reverse approach, when we immunoprecipitated the polyQ polypeptides with anti-GFP antibody and then blotted with antiubiquitin antibody, also gave negative results, suggesting that no significant ubiquitination of 103QP and other polyQ polypeptides occurs in yeast. There is a possibility that on ubiquitination the polyQ polypeptides are rapidly incorporated into aggregates containing all the ubiquitinated forms. Therefore, we subjected the lysates of cells coexpressing His-Ub with 103Q or 103QP polypeptides to differential centrifugation and immunoblotted the resulting fractions with anti-GFP antibody. No bands above a monomer of either 103Q or 103QP were detected with anti-GFP antibody (Supplemental Fig. 5B), further confirming the lack of significant ubiquitination of these polypeptides. Similar experiments showed that the polyQ polypeptides also do not undergo modification with other ubiquitin-like proteins, such as SUMO (Supplemental Fig. 5B).

There was a possibility that even minor undetectable ubiquitination could be sufficient for aggresome targeting. Therefore we decided to test whether the knockout of potential ubiquitination sites in 103QP affects aggresome formation. In huntingtin exon1 there are 3 lysines, all of which are located at the N-terminal 17-amino acid segment (N17). In Drosophila, 103Q and 103QP undergo ubiquitination and SUMOylation at 2 out of these 3 lysines (51). We substituted all 3 lysines (K6, K9 and K12) in 103QP with arginine and examined the effect on aggresome formation. Microscopic observation indicated that these substitutions did not affect aggresome formation (not shown). We cannot completely exclude a minor ubiquitination of lysines present in GFP. However, it is unlikely that this possible ubiquitination provides aggresome formation, because 103QP with other tags of entirely different sequence (e.g., RFP) is also targeted to an aggresome (see, e.g., Fig. 1B). Together these data suggest that neither ubiquitination nor SUMOylation of 103QP play a critical role in the aggresome targeting.

On the other hand, the aggresome formation was inhibited in a thermosensitive uba1 mutant with defective ubiquitin-activating enzyme E1 (Supplemental Fig. 6). Therefore, it seems likely that the process of aggresome formation involves ubiquitination of a critical cellular component, but not of a substrate.

The 14-3-3 protein Bmh1 is necessary for aggresome targeting

Among the proteins associated with both 103QP aggresomes and 103Q multiple nonaggresome aggregates we found a 14-3-3 protein Bmh1 (Table 2). This protein was of special interest because 14-3-3 proteins have been reported to associate with huntingtin and α-synuclein aggregates in mammalian cells (52, 53), but their role in protein aggregation remains unknown. Therefore, despite the ability of Bmh1 to bind both aggresomes and nonaggresomal aggregates (see Table 2 and Fig. 4A), we tested whether deletion of bmh1 can affect 103QP aggresome formation. Surprisingly, in the Δbmh1 mutant 103QP did not form an aggresome but rather multiple aggregates reminiscent of those of 103Q (Fig. 5A). This aggregation of 103QP was delayed compared with the wild type. In yeast there are only 2 closely homologous 14-3-3 proteins Bmh1 and Bmh2. Interestingly, bmh2 deletion did not affect 103QP aggresomes (not shown), indicating the lack of functional redundancy of these 14-3-3 proteins in the process of aggresome formation. Notably, bmh1 deletion also inhibited aggresome formation by 103Q expressed from MET promoter at low levels (Fig. S4). Therefore Bmh1 appears to be critical for the aggresome formation. Notably, bmh1 deletion not only blocked 103QP aggresome formation but also led to high toxicity of 103QP (Fig. 5B). These data further indicate that aggresome formation is protective, and 103QP becomes toxic if it cannot form an aggresome.

Figure 5.
Bmh1 is essential for aggresome formation. Fluorescent microscopy images of live cells. A) bmh1 deletion leads to formation of multiple aggregates by 103QP (right panel). Fluorescent microscopy images of live cells. B) bmh1 deletion causes toxicity of ...

To address the role of the 14-3-3 protein in the aggresome formation, we tested whether Bmh1 can associate with soluble polyQ molecules. Wild-type cells expressing 103QP, 103Q, or 25QP were subjected to homogenization and centrifugation at 12,000 g to clarify the lysates. The polypeptides were then immunoprecipitated with anti-FLAG antibody. As a control for nonspecific binding, we used cells transformed with an empty vector. The immunoprecipitated proteins were analyzed by immunoblotting with anti-Bmh1/Bmh2 antibody. As seen in Fig. 5C, Bmh1 coprecipitated with soluble 103QP and even to a higher extent with 103Q, whereas little Bmh1 was found in association with 25QP. This finding indicates that binding of Bmh1 to polyQ does not require the P-region, but binding depends on the length of polyQ. Therefore, Bmh1 probably mediates the aggresome formation by binding directly to polyQ oligomers. Because Bmh1 was found to bind independently of the P-region, its binding site has to be either the polyQ domain or the N17 region in front of it. To discern the Bmh1 binding domain we deleted the N17 region and tested whether the truncated 103QP can be coimmunoprecipitated with Bmh1. As shown in Fig. 5D, the N17 deletion completely abolished interaction of Bmh1 with soluble 103QP molecules, indicating that N17 is critical for association with Bmh1. Because Bmh1 binding was facilitated significantly by the polyQ expansion (Fig. 5C), the length of an adjacent polyQ domain is also important. In line with the importance of Bmh1 binding, aggresome formation was significantly retarded in the 103QP lacking N17 (ΔN17-103QP). Indeed, after 6 h induction, whereas 103QP formed a typical aggresome, ΔN17-103QP either remained soluble or formed multiple small aggregates (Fig. 5E) similar to those formed by 103QP in Δbmh1 cells (Fig. 5A). It should be noted that the effect of bmh1 deletion on aggresome formation was more profound than that of N17 deletion, which prevents binding of Bmh1 to 103QP. After prolonged expression (overnight), ΔN17-103QP aggresomes could be seen in some cells, while still no 103QP aggresomes were formed in bmh1 cells. These data suggest that the delayed aggresome formation by the ΔN17-103QP polypeptide could be due to a slow recruitment into polyQ oligomers of polyQ-containing endogenous proteins that in turn can recruit Bmh1.

The data presented above indicate that Bmh1 does not discriminate between 103Q and 103QP aggregates and strongly associates with their soluble forms, which suggests that this 14-3-3 protein functions earlier than Cdc48 in the aggresome-targeting pathway. Therefore, we investigated whether binding to Bmh1 is important for recruitment of Cdc48 (VCP/p97) into 103QP aggregates. Accordingly, we introduced bmh1 deletion into the strain with endogenous Cdc48-GFP. In this mutant we expressed 103QP-RFP and monitored by fluorescent microscopy the association of Cdc48-GFP with 103QP-RFP aggregates. No significant association of Cdc48-GFP with 103QP-RFP aggregates was seen in these cells (Fig. 4F), in contrast to its colocalization with 103QP-RFP aggresome that was detected in the corresponding wild-type strain. This observation strongly suggests that Bmh1 works upstream of Cdc48 (VCP/p97) in the aggresome targeting pathway and is essential for the Cdc48 recruitment.

DISCUSSION

The aggresome was first described as a novel organelle serving as a depot of protein aggregates colocalized with centrosome that is formed in a microtubule-dependent fashion (7). However, other protein aggregation pathways also exist in cells. To elucidate mechanisms of the aggresome pathway, it is critical to define it using mechanism-based criteria. This crucial constraint is often missed in the literature when investigators study undefined formation of “protein aggregates” or “inclusion bodies.” In the present study, we reinforced the original definition of the aggresome using critical mechanism-based criteria and subsequently discovered that yeast possesses an aggresome machinery.

In yeast, 103QP aggregates represent true aggresomes, because their formation requires microtubule-dependent transport (Fig. 1B), and they associate with the centrosome (SPB in yeast) (Fig. 1C and Supplemental Fig. 1B). Such yeast aggresomes resemble mammalian aggresomes in the sense that certain components of the ubiquitin-proteasome machinery and chaperones are recruited into this structure (Table 2). We could not observe transport of small aggregates to an aggresome, probably because they are too small to be detected by microscopy; our coexpression experiments clearly indicated that polyQ polypeptides can be transported to an aggresome in the form of oligomers. These findings suggest that the aggresome machinery is conserved in evolution, and hence yeast could provide a valid model for elucidating mechanisms of aggresome formation in mammalian cells.

Our results underscore that aggresome formation is an important survival mechanism that may allow cells to withstand a buildup of abnormal polypeptides. Previous reports have indicated that aggresome formation correlates with cell survival, and that inhibition of microtubules enhances proteotoxic effects (8, 52). Data presented here stress the significance of aggresome targeting for relieving the polyQ toxicity, because forcing a toxic protein to an aggresome, for example, in 25QP/103Q mixing experiment, relieves the toxicity (Supplemental Fig. 2). Moreover, preventing aggresome formation by various mutations, such as cdc48 or bmh1, makes 103QP toxic (Figs. 4D and and5B).5B). Of note, soluble 25QP did not cause toxicity in these mutants. Therefore, chaperones and UPS appear to serve as first lines of cellular defense, but, if they fail to prevent accumulation of misfolded polypeptides, the aggresome machinery is activated as the last resort. In mammalian cells, aggresome formation by various polypeptides is usually initiated in response to proteasome failure, such as on addition of proteasome inhibitors (21, 54).

A novel concept that emerges from this study is that targeting to an aggresome is determined by specific domains in substrates. With huntingtin exon 1, this signal is the proline-rich P-region that is located immediately downstream of polyQ. Certainly the P-region is not the only aggresome-targeting signal. For example, we have found that the protein synphilin 1 that plays a role in Parkinson’s disease also has a distinct aggresome-targeting signal with no homology with the P-region (55). In that study we demonstrated that aggresome-targeting signals are transferable. Indeed, fusion with 25QP can facilitate aggresome formation by synphilin 1 fragment lacking an endogenous aggresome-targeting signal and vice versa, fusion with the aggresome-targeting domain of synphilin 1 can facilitate aggresome formation by 103Q.

Becaue aggresome-targeting domains of huntingtin and synphilin 1 are so diverse, there should be diverse recognition factors for these signals. Such a system would, in a way, resemble the ubiquitination machinery, where various degradation substrates have diverse ubiquitination signals that are recognized by diverse ubiquitin ligases E3s. On the other hand, proline-rich domains could play a role in aggresome targeting of many substrates. Mammalian proteins that have polyQ domains quite often (more than 30%) contain defined proline-rich domains in the vicinity. It is possible that proteins with polyQ domains have a high tendency to aggregate, and if aggregation happens, adjacent proline-rich domains would serve to protect cells by facilitating aggresome formation.

We found several molecular factors involved in aggresome formation. These findings were initiated by analysis of purified 103Q and 103QP aggregates by mass spectrometry, which identified a number of associated proteins. We tested for their involvement in aggresome formation. Some protein deletions of the corresponding genes had mild effects; for example, sse1 and hsp42 deletions slowed down aggregation (data not shown). Only the mutation in Cdc48 or deletion of Bmh1 caused severe 103QP aggresome disruption. Moreover, Bmh1 and Cdc48 appear to play a role in the low-capacity P-region-independent aggresome targeting as well, because mutations in the corresponding genes disrupted aggresomes and caused toxicity of 103Q expressed at low levels (not shown). Cdc48 is an abundant, highly conserved AAA ATPase involved in a variety of important cellular processes, including ubiquitin-mediated protein degradation, mitotic spindle disassembly, and Golgi transport (56, 57). The heterodimeric Ufd1/Npl4 cofactor is required for Cdc48 function in the ubiquitin/proteasome pathways, including ERAD and UFD (58, 59). Analysis of 103QP aggregation in the corresponding mutants demonstrated that both Ufd1 and Nlp4 are important for aggresome formation (Fig. 4E), though other Cdc48-associated proteins did not affect aggresome formation. The possible involvement of the mammalian Cdc48 homologue VCP/p97, as well as Ufd1 and Nlp4, in formation of an aggresome by a bulk of misfolded endogenous proteins has been reported previously (60). Here we describe the role of these proteins in aggresome formation of polyQ polypeptides. These data complement the findings of M. Duennwald and S. Lindquist (unpublished results) that Cdc48, Ufd1, and Nlp4 relieve the ER stress caused by polyQ and suppress the polyQ toxicity.

The known role of Cdc48 and its cofactors in UPS suggest that these proteins participate in an ubiquitination reaction that is critical for aggresome formation. However, we demonstrated that neither 103QP nor 103Q undergoes ubiquitination in yeast. The fact that aggresome formation is blocked in the E1 ubiquitin-activating enzyme uba1-204 mutant at nonpermissive temperature (Supplemental Fig. 6) indicates that aggresome formation may require ubiquitination of certain components of the aggresome machinery.

Interestingly, Bmh1 associated with soluble forms of both 103Q and 103QP and remained in the complexes through the entire aggregation processes ending up in both multiple 103Q aggregates and the 103QP aggresome. Interaction of Bmh1 with polyQ polypeptides was independent on the P-region but required the N17 domain. Cdc48 recruitment appears to be the P-region-specific, and so Cdc48 was found associated predominantly with the 103QP aggresome. Furthermore, Bmh1 was critical for such recruitment because Cdc48-GFP did not colocalize with multiple 103QP aggregates seen in bmh1 mutant (Fig. 4F). Accordingly, there might be a P-region recognition factor that binds to 103QP when it is already in a complex with Bmh1 and further recruits Cdc48.

Clearly there might be additional components of the aggresome-sorting machinery. Identification of further components using the yeast model has the potential to uncover important details of the mechanisms of this powerful protective system.

Acknowledgments

This work was supported by U.S. National Institutes of Health grants R01 NS047705 (to M.Y.S.), P41 RR10888 and S10 RR15942 (to C.E.C.), and R01GM58763 (to Y.O.C.). We are grateful to Dr. Martin Duennwald (Boston Biomedical Research Institute, Boston, MA, USA) for very helpful suggestions and insights.

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