Abnormal proteins can form aggresome in yeast: aggresome-targeting signals and components of the machinery.
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.
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, USA.
Yan
Y
Wang
Anatoli B
AB
Meriin
Nava
N
Zaarur
Nina V
NV
Romanova
Yury O
YO
Chernoff
Catherine E
CE
Costello
Michael Y
MY
Sherman
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity; D054875:Ubiquitination; D000074405:Valosin Containing Protein
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C097188:polyglutamine; D000251:Adenosine Triphosphatases; C000614177:CDC48 protein, S cerevisiae; D000074405:Valosin Containing Protein