Glycine-alanine repeats impair proper substrate unfolding by the proteasome.
Proteasome ATPases unravel folded proteins. Introducing a sequence containing only glycine and alanine residues (GAr) into substrates can impair their digestion. We previously proposed that a GAr interferes with the unfolding capacity of the proteasome, leading to partial degradation of products. Here we tested that idea in several ways. Stabilizing or destabilizing a folded domain within substrate proteins changed GAr-mediated intermediate production in the way predicted by the model. A downstream folded domain determined the sites of terminal proteolysis. The spacing between a GAr and a folded domain was critical for intermediate production. Intermediates containing a GAr did not remain associated with proteasomes, excluding models whereby retained GAr-containing proteins halt further processing. The following model is supported: a GAr positioned within the ATPase ring reduces the efficiency of coupling between nucleotide hydrolysis and work performed on the substrate. If this impairment takes place when unfolding must be initiated, insertion pauses and proteolysis is limited to the portion of the substrate that has already entered the catalytic chamber of the proteasome.
Hoyt
Martin A
MA
Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0414, USA.
Zich
Judith
J
Takeuchi
Junko
J
Zhang
Mingsheng
M
Govaerts
Cedric
C
Coffino
Philip
P
D000409:Alanine / Q000235:genetics*; D000818:Animals; D005998:Glycine / Q000235:genetics*; D006868:Hydrolysis; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D046988:Proteasome Endopeptidase Complex; D009955:Ornithine Decarboxylase; D000409:Alanine; D005998:Glycine