Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits

Abstract

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/ CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1␣/PFD5 as a novel c-Myc-binding protein and found that MM-1␣ suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1␣ into cells increased the level of endogenous MM-1␣. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1␣ but not that of endogenous MM-1␣, indicating that overexpressed MM-1␣, but not endogenous MM-1␣, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after cotransfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. Molecular chaperones assist newly synthesized or denatured proteins to be naturally folded in cells (1). Prefoldin (PFD)2 was identified as an unfolded actin-binding protein (2) and was shown to play a role as a chaperone by recruiting substrates such as actins and tubulins to the eukaryotic cytosolic chaperonin TRiC/CCT, which facilitates the folding of proteins through ATP hydrolysis (3-6). It has been reported that deletion of genes encoding prefoldin subunits in yeast resulted in cytoskeletal defects and in a slow-growth phenotype (2, 7, 8), which are similar to those found in mutations in CCT subunits, suggesting that coordinated function of prefoldin and TRiC/ CCT is necessary for cells to maintain normal cytoskeletons.Elucidation of the crystal structure of Methanobacterium thermoautotrophicum prefoldin (9) and results of analysis of eukaryotic prefoldin by electron microscopy (10) revealed that prefoldin is a hexameric complex with a jellyfish-like structure and that each subunit forms a coiled-coil structure at the Nand C-terminal ␣ helices. Archaea prefoldin possesses only two subunits, prefoldin ␣ and ␤, and builds up a ␣2␤4 hexamer (11). Eukaryotic prefoldin, on the other hand, possess six subunits, two ␣ subunits (PFD3 and PFD5) and four ␤ subunits (PFD1, PFD2, PFD4, and PFD6) (9, 11). In both archaea and eukaryotes, prefoldin ␣ subunits contain two ␤ hairpins in connecting regions at both terminals, and the ␤ subunits contain one ␤ hairpin to a...

Prefoldin Protects Neuronal Cells from Polyglutamine Toxicity by Preventing Aggregation Formation

“…1B, amounts of the prefoldin complex in cells that had been transfected with PFD2 and PFD5 siRNAs were reduced to 12 and 15%, respectively, of that in control cells, indicating that RNAi-mediated knockdown of PFD2 and PFD5 effectively reduced formation of the prefoldin complex. As we reported previously (38), these results suggest that free forms of prefoldin subunits are unstable compared with prefoldin subunits in the prefoldin complex.…”

“…In addition to reduced levels of PFD2 and PFD5, protein expression levels of other subunits were also reduced, although the levels of PFD2 and PFD3 were unaffected by PFD5 siRNA. These results support the notion that the expression levels of prefoldin subunits are mutually regulated at the protein level through the ubiquitin-proteasome system as we reported previously (38). To examine whether knockdown of a prefoldin subunit affects formation of the prefoldin complex, proteins extracted from siRNA-transfected cells were separated on a glycerol density gradient followed by Western blotting with anti-PFD2 and anti-PFD5 antibodies.…”

“…Toxic soluble oligomers are thought to exert their toxicity by interacting with other proteins (e.g. coaggregation with functional cellular proteins such as transcription factors TBP and CBP) (38). Co-existence of nontoxic and toxic oligomers of amyloidogenic proteins in cells has indeed been reported (45).…”

“…hydrophobic ␤-sheets) cannot easily be changed into nontoxic forms, prefoldin probably transfers them to CCT or other appropriate chaperones. Prefoldin might also deliver misfolded toxic species to the proteasome for degradation through direct interaction (38,46).…”

Common Mechanisms of Onset of Cancer and Neurodegenerative Diseases

“…Knockdown of MM-1/PFD5 resulted in disruption of the prefoldin complex and loss of protective activity against oligomer formation of pathogenic Huntingtin and α-synuclein. [37][38][39] Prefoldin also modulates aggregation of Amyloid-β (Aβ). 40,41) Furthermore, mice possessing a missense mutation in MM-1α at amino acid number 110 from leucine to arginine (L110R) exhibit various symptoms, including degeneration of the cerebellum and retina and male infertility.…”