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© The Rockefeller University Press, 0021-9525/2001//283 $5.00
The Journal of Cell Biology, Volume 153, Number 2, , 2001 283-294

Identities of Sequestered Proteins in Aggregates from Cells with Induced Polyglutamine Expression

^a Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037
^b Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, California 90095
^c Departments of Chemistry and Biochemistry, University of California, Los Angeles, California 90095
^d Departments of Psychiatry and Biobehavioral Sciences and the Neuropsychiatric Institute, University of California, Los Angeles, California 90095
The Salk Institute for Biological Studies, 10010 North Torrey Pines Rd., La Jolla, CA, 92037.(858) 453-4100 ext

Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle–regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

Key Words: polyglutamine • Huntington's disease • aggregates • inducible expression • ecdysone receptor

© 2001 The Rockefeller University Press

Abbreviations used in this paper: CMV, cytomegalovirus; GFP, green fluorescent protein; HD, Huntington's disease; Htt, Huntingtin; IA, intracellular aggregate; MALDI, matrix-assisted laser desorption ionization time of flight mass spectrometry; MEF-2a, myocyte-specific enhancer factor; NDST, normal donkey serum and Triton X-100; NFL, light neurofilament protein; NLS, nuclear localization signal; NPC, nuclear pore complex; NPCP, NPC protein; polyP, polyproline; polyQ, polyglutamine; TatBP-1, Tat-binding protein-1; TBP, TATA-binding protein; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP–biotin end labeling.

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