{"format":"molecular-machines.cards","version":1,"updated":"2026-10-01","decks":[{"machine":"proteasome","name":"Proteasome","count":28,"url":"/learn/cards/proteasome.json"}],"cards":[{"id":"proteasome-tag-signal","machine":"proteasome","kind":"qa","prompt":"What mark does the cell put on a protein to send it to the 26S proteasome?","answer":"A chain of ubiquitin.","explanation":"Ubiquitin receptors in the proteasome bind this chain, so the proteasome picks marked proteins out of thousands of others.","section":"summary","topic":"purpose","sources":["machine:summary","step:A tagged protein arrives","ref:dong2019"],"tags":["ubiquitin","recognition"],"difficulty":1,"url":"/machines/proteasome#summary","cites":[{"source":"machine:summary","machine":"proteasome","label":"Summary","section":"summary"},{"source":"step:A tagged protein arrives","machine":"proteasome","label":"Step: A tagged protein arrives","section":"mechanism"},{"source":"ref:dong2019","machine":"proteasome","label":"Dong et al.","section":"sources","anchor":"ref-dong2019","href":"https://doi.org/10.1038/s41586-018-0736-4"}]},{"id":"proteasome-sealed-barrel-why","machine":"proteasome","kind":"qa","prompt":"Why does the proteasome keep its cutting sites inside a sealed barrel?","answer":"So that only a chain fed through the gate meets them, which keeps the cell's other proteins safe.","explanation":"All six active sites face the inner chamber, and the gate into it is shut until the ATPase ring opens it.","section":"summary","topic":"purpose","sources":["step:Cut into short peptides","stop:barrel","ref:groll1997"],"tags":["core-particle","safety"],"difficulty":2,"url":"/machines/proteasome#summary","cites":[{"source":"step:Cut into short peptides","machine":"proteasome","label":"Step: Cut into short peptides","section":"mechanism"},{"source":"stop:barrel","machine":"proteasome","label":"Big picture: Inside the barrel","section":"story"},{"source":"ref:groll1997","machine":"proteasome","label":"Groll et al.","section":"sources","anchor":"ref-groll1997","href":"https://doi.org/10.1038/386463a0"}]},{"id":"proteasome-atpase-ring-role","machine":"proteasome","kind":"qa","prompt":"Which part of the 26S proteasome grips, unfolds and pulls the tagged protein into the barrel?","answer":"The ring of six AAA+ ATPases, Rpt1 to Rpt6.","explanation":"The same ring also opens the gate into the core particle.","section":"summary","topic":"parts","sources":["component:Rpt1-Rpt6","stat:ATPase subunits","ref:lander2012"],"tags":["atpase","motor"],"difficulty":1,"url":"/machines/proteasome#summary","cites":[{"source":"component:Rpt1-Rpt6","machine":"proteasome","label":"Part: Rpt1-Rpt6","section":"summary"},{"source":"stat:ATPase subunits","machine":"proteasome","label":"Key number: ATPase subunits","section":"summary"},{"source":"ref:lander2012","machine":"proteasome","label":"Lander et al.","section":"sources","anchor":"ref-lander2012","href":"https://doi.org/10.1038/nature10774"}]},{"id":"proteasome-rpn11-role","machine":"proteasome","kind":"qa","prompt":"What does Rpn11, a zinc enzyme of the proteasome lid, do to the substrate?","answer":"It cuts the ubiquitin chain off the substrate at the pore entrance.","explanation":"Rpn11 is a deubiquitinase. It removes the tag in the same cycle that pulls the protein in.","section":"summary","topic":"parts","sources":["component:Rpn11","mechanism:Deubiquitination","ref:delapena2018"],"tags":["rpn11","ubiquitin"],"difficulty":1,"url":"/machines/proteasome#summary","cites":[{"source":"component:Rpn11","machine":"proteasome","label":"Part: Rpn11","section":"summary"},{"source":"mechanism:Deubiquitination","machine":"proteasome","label":"Step: Deubiquitination","section":"mechanism"},{"source":"ref:delapena2018","machine":"proteasome","label":"de la Pena et al.","section":"sources","anchor":"ref-delapena2018","href":"https://doi.org/10.1126/science.aav0725"}]},{"id":"proteasome-active-beta-subunits","machine":"proteasome","kind":"qa","prompt":"Which three beta subunits of the proteasome core particle carry the active sites?","answer":"beta1, beta2 and beta5.","explanation":"They cut after acidic, basic and bulky water-repelling residues: caspase-like, trypsin-like and chymotrypsin-like. The other four beta types are structural.","section":"summary","topic":"parts","sources":["component:beta1, beta2, beta5","step:Cut into short peptides","ref:groll1997"],"tags":["core-particle","active-site"],"difficulty":2,"url":"/machines/proteasome#summary","cites":[{"source":"component:beta1, beta2, beta5","machine":"proteasome","label":"Part: beta1, beta2, beta5","section":"summary"},{"source":"step:Cut into short peptides","machine":"proteasome","label":"Step: Cut into short peptides","section":"mechanism"},{"source":"ref:groll1997","machine":"proteasome","label":"Groll et al.","section":"sources","anchor":"ref-groll1997","href":"https://doi.org/10.1038/386463a0"}]},{"id":"proteasome-core-subunits","machine":"proteasome","kind":"cloze","prompt":"The proteasome core particle has 28 subunits in {{four rings of seven}}: (alpha1-7 beta1-7)2.","answer":"four rings of seven","explanation":"Two outer alpha rings form the gate; two inner beta rings hold the active sites, three per ring, so six sites per core.","section":"summary","topic":"numbers","sources":["stat:Core particle subunits","stat:Catalytic sites per core","ref:groll1997"],"tags":["core-particle","structure"],"difficulty":2,"url":"/machines/proteasome#summary","cites":[{"source":"stat:Core particle subunits","machine":"proteasome","label":"Key number: Core particle subunits","section":"summary"},{"source":"stat:Catalytic sites per core","machine":"proteasome","label":"Key number: Catalytic sites per core","section":"summary"},{"source":"ref:groll1997","machine":"proteasome","label":"Groll et al.","section":"sources","anchor":"ref-groll1997","href":"https://doi.org/10.1038/386463a0"}]},{"id":"proteasome-hbyx-older","machine":"proteasome","kind":"qa","prompt":"Why is the proteasome's HbYX gate signal thought to be older than the 26S particle itself?","answer":"Archaea use the simpler PAN ATPase with the same HbYX gate signal as the eukaryotic Rpt subunits.","explanation":"Free HbYX peptides of 7 to 10 residues open the gate of the archaeal core on their own (Smith et al. 2007).","section":"summary","topic":"parts","sources":["evolution:AAA+ origin of the ATPase ring","ref:smith2007","ref:rabl2008"],"tags":["evolution","gate"],"difficulty":3,"url":"/machines/proteasome#summary","cites":[{"source":"evolution:AAA+ origin of the ATPase ring","machine":"proteasome","label":"AAA+ origin of the ATPase ring","section":"summary"},{"source":"ref:smith2007","machine":"proteasome","label":"Smith et al.","section":"sources","anchor":"ref-smith2007","href":"https://doi.org/10.1016/j.molcel.2007.06.033"},{"source":"ref:rabl2008","machine":"proteasome","label":"Rabl et al.","section":"sources","anchor":"ref-rabl2008","href":"https://doi.org/10.1016/j.molcel.2008.03.004"}]},{"id":"proteasome-protac","machine":"proteasome","kind":"qa","prompt":"How does a PROTAC get the proteasome to destroy a chosen protein?","answer":"It ties the protein to an E3 ligase, which marks it with ubiquitin.","explanation":"The two-headed molecule supplies the address; the proteasome does the rest. dBET1 removed BET proteins in cells and in mice (status: demonstrated).","section":"summary","topic":"debate","sources":["frontier:Targeted degradation with PROTACs","ref:winter2015","ref:sakamoto2001"],"tags":["protac","drugs"],"difficulty":2,"url":"/machines/proteasome#summary","cites":[{"source":"frontier:Targeted degradation with PROTACs","machine":"proteasome","label":"Open question: Targeted degradation with PROTACs","section":"summary"},{"source":"ref:winter2015","machine":"proteasome","label":"Winter et al.","section":"sources","anchor":"ref-winter2015","href":"https://doi.org/10.1126/science.aab1433"},{"source":"ref:sakamoto2001","machine":"proteasome","label":"Sakamoto et al.","section":"sources","anchor":"ref-sakamoto2001","href":"https://doi.org/10.1073/pnas.141230798"}]},{"id":"proteasome-commitment","machine":"proteasome","kind":"qa","prompt":"Ubiquitin binding alone does not commit a protein to the proteasome. What does?","answer":"The pore loops of the ATPase ring gripping the protein's unstructured tail.","explanation":"Ubiquitin chains bind and leave quickly. Only a substrate whose tail engages the motor triggers the switch to the working shape and goes on to be degraded.","section":"mechanism","topic":"cycle","sources":["step:The loose tail enters the pore","step:A tagged protein arrives","ref:bard2019"],"tags":["engagement","tail"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"step:The loose tail enters the pore","machine":"proteasome","label":"Step: The loose tail enters the pore","section":"mechanism"},{"source":"step:A tagged protein arrives","machine":"proteasome","label":"Step: A tagged protein arrives","section":"mechanism"},{"source":"ref:bard2019","machine":"proteasome","label":"Bard et al.","section":"sources","anchor":"ref-bard2019","href":"https://doi.org/10.1016/j.cell.2019.02.031"}]},{"id":"proteasome-pull-speeds-cut","machine":"proteasome","kind":"cloze","prompt":"Rpn11 cuts the ubiquitin chain off faster when {{the motor pulls on the substrate}}.","answer":"the motor pulls on the substrate","explanation":"This couples tag removal to translocation: the tag comes off as the protein moves into the pore.","section":"mechanism","topic":"cycle","sources":["mechanism:Deubiquitination","step:Rpn11 cuts off the tag","ref:delapena2018"],"tags":["rpn11","coupling"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Deubiquitination","machine":"proteasome","label":"Step: Deubiquitination","section":"mechanism"},{"source":"step:Rpn11 cuts off the tag","machine":"proteasome","label":"Step: Rpn11 cuts off the tag","section":"mechanism"},{"source":"ref:delapena2018","machine":"proteasome","label":"de la Pena et al.","section":"sources","anchor":"ref-delapena2018","href":"https://doi.org/10.1126/science.aav0725"}]},{"id":"proteasome-gate-opening","machine":"proteasome","kind":"qa","prompt":"How do the proteasome's ATPases open the gate into the core particle?","answer":"Their C-terminal HbYX tails dock in pockets between alpha subunits, which rotates the alpha subunits and opens the gate.","explanation":"The tails work like a key in a lock. Closed, the N-terminal tails of the alpha subunits fill the entrance.","section":"mechanism","topic":"cycle","sources":["mechanism:Gate opening","step:The gate opens","ref:smith2007"],"tags":["gate","hbyx"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Gate opening","machine":"proteasome","label":"Step: Gate opening","section":"mechanism"},{"source":"step:The gate opens","machine":"proteasome","label":"Step: The gate opens","section":"mechanism"},{"source":"ref:smith2007","machine":"proteasome","label":"Smith et al.","section":"sources","anchor":"ref-smith2007","href":"https://doi.org/10.1016/j.molcel.2007.06.033"}]},{"id":"proteasome-staircase-one-way","machine":"proteasome","kind":"qa","prompt":"The six proteasome ATPases stand in a spiral staircase. Why does the substrate chain move one way only?","answer":"ATP hydrolysis passes around the ring in order, so each subunit grips the chain in turn.","explanation":"The gripping subunits move down together and carry the chain with them, hand over hand.","section":"mechanism","topic":"cycle","sources":["mechanism:Translocation in steps","step:Hand over hand","ref:dong2019"],"tags":["atpase","translocation"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Translocation in steps","machine":"proteasome","label":"Step: Translocation in steps","section":"mechanism"},{"source":"step:Hand over hand","machine":"proteasome","label":"Step: Hand over hand","section":"mechanism"},{"source":"ref:dong2019","machine":"proteasome","label":"Dong et al.","section":"sources","anchor":"ref-dong2019","href":"https://doi.org/10.1038/s41586-018-0736-4"}]},{"id":"proteasome-seam-subunit","machine":"proteasome","kind":"qa","prompt":"In the proteasome's ATPase staircase, what does the subunit at the bottom do next?","answer":"It lets go of the chain and climbs to the top.","explanation":"This 'seam' subunit then binds ATP and grips the chain again at the top, so the ring keeps cycling.","section":"mechanism","topic":"cycle","sources":["step:Hand over hand","evidence:substrate-engaged-motor-states","ref:delapena2018"],"tags":["atpase","translocation"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"step:Hand over hand","machine":"proteasome","label":"Step: Hand over hand","section":"mechanism"},{"source":"evidence:substrate-engaged-motor-states","machine":"proteasome","label":"Motor states seen in the substrate-engaged yeast 26S proteasome (de la Peña AH 2018)","section":"evidence","anchor":"ev-substrate-engaged-motor-states"},{"source":"ref:delapena2018","machine":"proteasome","label":"de la Pena et al.","section":"sources","anchor":"ref-delapena2018","href":"https://doi.org/10.1126/science.aav0725"}]},{"id":"proteasome-hinge","machine":"proteasome","kind":"cloze","prompt":"In each proteasome ATPase, a {{hinge motion}} driven by ATP hydrolysis sets when that subunit holds or releases the chain.","answer":"hinge motion","explanation":"Bound nucleotide locks the large and small AAA+ subdomains into one rigid body; release lets them hinge by 20–25°.","section":"mechanism","topic":"cycle","sources":["mechanism:Unfolding","step:Hand over hand","ref:dong2019"],"tags":["atpase","unfolding"],"difficulty":3,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Unfolding","machine":"proteasome","label":"Step: Unfolding","section":"mechanism"},{"source":"step:Hand over hand","machine":"proteasome","label":"Step: Hand over hand","section":"mechanism"},{"source":"ref:dong2019","machine":"proteasome","label":"Dong et al.","section":"sources","anchor":"ref-dong2019","href":"https://doi.org/10.1038/s41586-018-0736-4"}]},{"id":"proteasome-thr1","machine":"proteasome","kind":"qa","prompt":"Which residue in the proteasome's active beta subunits attacks the peptide bond?","answer":"The N-terminal threonine (Thr1).","explanation":"It sits on beta1, beta2 and beta5. Deleting Thr1 or changing it to alanine stops the enzyme.","section":"mechanism","topic":"cycle","sources":["mechanism:Cutting","stat:Catalytic residue","ref:seemuller1995"],"tags":["active-site","threonine"],"difficulty":1,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Cutting","machine":"proteasome","label":"Step: Cutting","section":"mechanism"},{"source":"stat:Catalytic residue","machine":"proteasome","label":"Key number: Catalytic residue","section":"summary"},{"source":"ref:seemuller1995","machine":"proteasome","label":"Seemueller et al.","section":"sources","anchor":"ref-seemuller1995","href":"https://doi.org/10.1126/science.7725107"}]},{"id":"proteasome-release","machine":"proteasome","kind":"qa","prompt":"What decides when peptides leave the proteasome barrel, according to Kisselev and colleagues' proposal?","answer":"Cutting goes on until a piece is short enough to diffuse out.","explanation":"Product length hardly changed when one type of active site was blocked. The products are 3 to 22 residues long.","section":"mechanism","topic":"cycle","sources":["mechanism:Release","step:Cut into short peptides","ref:kisselev1999"],"tags":["peptides","release"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Release","machine":"proteasome","label":"Step: Release","section":"mechanism"},{"source":"step:Cut into short peptides","machine":"proteasome","label":"Step: Cut into short peptides","section":"mechanism"},{"source":"ref:kisselev1999","machine":"proteasome","label":"Kisselev et al.","section":"sources","anchor":"ref-kisselev1999","href":"https://doi.org/10.1074/jbc.274.6.3363"}]},{"id":"proteasome-body-turnover","machine":"proteasome","kind":"cloze","prompt":"An adult human breaks down and rebuilds about {{300–400 g}} of body protein a day, but eats only 50–80 g.","answer":"300–400 g","explanation":"Most of the parts are recycled, and most of that breakdown happens in the proteasome. The figure is a review estimate.","section":"story","topic":"purpose","sources":["fact:P1","stop:body","ref:schutz2011"],"tags":["body","turnover"],"difficulty":2,"url":"/machines/proteasome#story","cites":[{"source":"fact:P1","machine":"proteasome","label":"Protein broken down and rebuilt per day: 300–400 g per day","section":"story"},{"source":"stop:body","machine":"proteasome","label":"Big picture: You, renewing","section":"story"},{"source":"ref:schutz2011","machine":"proteasome","label":"Schutz 2011","section":"sources","href":"https://doi.org/10.1024/0300-9831/a000064"}]},{"id":"proteasome-pieces-fate","machine":"proteasome","kind":"qa","prompt":"Most proteasome peptides become amino acids for new proteins. What happens to a few of them?","answer":"They go to the cell surface on MHC class I, where immune cells check them.","explanation":"The display is a sample of what the cell is making, so immune cells can spot an infected cell.","section":"story","topic":"purpose","sources":["stop:pieces","fact:P8-mhc","ref:schubert2000"],"tags":["immune","mhc"],"difficulty":1,"url":"/machines/proteasome#story","cites":[{"source":"stop:pieces","machine":"proteasome","label":"Big picture: The pieces","section":"story"},{"source":"fact:P8-mhc","machine":"proteasome","label":"Length of the peptides shown on MHC class I: 8–10 amino acids","section":"story"},{"source":"ref:schubert2000","machine":"proteasome","label":"Schubert 2000","section":"sources","href":"https://doi.org/10.1038/35008096"}]},{"id":"proteasome-vs-ribosome-rate","machine":"proteasome","kind":"qa","prompt":"In mouse L929 cells, how does the number of proteins the proteasomes destroy per minute compare with the number the ribosomes make?","answer":"About half: 2 × 10^6 destroyed versus 4 × 10^6 made per minute.","explanation":"It is a calculation: 8 × 10^5 proteasomes × 2.5 proteins per minute each. Together the two machines set protein levels.","section":"story","topic":"numbers","sources":["fact:P12","fact:P11","link:ribosome","ref:princiotta2003"],"tags":["cell","ribosome"],"difficulty":3,"url":"/machines/proteasome#story","cites":[{"source":"fact:P12","machine":"proteasome","label":"Proteins destroyed per minute in one mouse cell: 2 × 10^6 per minute","section":"story"},{"source":"fact:P11","machine":"proteasome","label":"Proteasomes in one mouse cell: 8 × 10^5 proteasomes","section":"story"},{"source":"link:ribosome","machine":"proteasome","label":"Link to Ribosome","section":"story"},{"source":"ref:princiotta2003","machine":"proteasome","label":"Princiotta 2003","section":"sources","href":"https://doi.org/10.1016/s1074-7613(03)00051-7"}]},{"id":"proteasome-shredder-breaks","machine":"proteasome","kind":"qa","prompt":"Where does the analogy 'the proteasome is a paper shredder' break down about how input gets in?","answer":"A shredder is fed; the proteasome pulls and unfolds its input itself, with six ATP motors.","explanation":"It also takes off the ubiquitin tag and returns it for reuse. The analogy gets right that the enclosed blades hurt nothing else.","section":"story","topic":"purpose","sources":["analogy:A paper shredder with a doorman"],"tags":["analogy"],"difficulty":2,"url":"/machines/proteasome#story","cites":[{"source":"analogy:A paper shredder with a doorman","machine":"proteasome","label":"Analogy: A paper shredder with a doorman","section":"story"}]},{"id":"proteasome-healthy-proteins","machine":"proteasome","kind":"qa","prompt":"Unlike a recycling plant, the proteasome also destroys healthy proteins on schedule. What does that let the cell do?","answer":"Turn signals off.","explanation":"For example, the proteasome destroys transcription factors, so it helps decide when genes switch on.","section":"story","topic":"purpose","sources":["analogy:A recycling plant","link:rna-polymerase"],"tags":["analogy","signalling"],"difficulty":2,"url":"/machines/proteasome#story","cites":[{"source":"analogy:A recycling plant","machine":"proteasome","label":"Analogy: A recycling plant","section":"story"},{"source":"link:rna-polymerase","machine":"proteasome","label":"Link to RNA polymerase II","section":"story"}]},{"id":"proteasome-time-per-protein","machine":"proteasome","kind":"cloze","prompt":"One mouse 26S proteasome needs about {{13 s}} to degrade one ubiquitinated DHFR molecule (Ub5-DHFR).","answer":"13 s","explanation":"A longer ubiquitinated protein, Sic1, took about twice as long (26 s), so time depends on the substrate.","section":"evidence","topic":"numbers","sources":["evidence:degradation-time-ub5-dhfr","stat:Time to degrade one tagged protein","ref:peth2013"],"tags":["rate","dhfr"],"difficulty":2,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:degradation-time-ub5-dhfr","machine":"proteasome","label":"Time for one 26S proteasome to degrade one ubiquitinated DHFR molecule (Peth A 2013)","section":"evidence","anchor":"ev-degradation-time-ub5-dhfr"},{"source":"stat:Time to degrade one tagged protein","machine":"proteasome","label":"Key number: Time to degrade one tagged protein","section":"summary"},{"source":"ref:peth2013","machine":"proteasome","label":"Peth et al.","section":"sources","anchor":"ref-peth2013","href":"https://doi.org/10.1074/jbc.m113.482570"}]},{"id":"proteasome-time-method","machine":"proteasome","kind":"qa","prompt":"How did Peth and colleagues get the 13 s a proteasome needs per Ub5-DHFR molecule?","answer":"From the maximal degradation rate (Vmax) at rising substrate levels: time per molecule = 1/Vmax.","explanation":"They followed 32P-labelled substrate turning into acid-soluble peptides with purified mouse 26S proteasomes; the maximum was about 4.7 molecules per minute per particle.","section":"evidence","topic":"numbers","sources":["evidence:degradation-time-ub5-dhfr","ref:peth2013"],"tags":["method","kinetics"],"difficulty":3,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:degradation-time-ub5-dhfr","machine":"proteasome","label":"Time for one 26S proteasome to degrade one ubiquitinated DHFR molecule (Peth A 2013)","section":"evidence","anchor":"ev-degradation-time-ub5-dhfr"},{"source":"ref:peth2013","machine":"proteasome","label":"Peth et al.","section":"sources","anchor":"ref-peth2013","href":"https://doi.org/10.1074/jbc.m113.482570"}]},{"id":"proteasome-atp-cost","machine":"proteasome","kind":"cloze","prompt":"Mouse 26S proteasomes spend {{50–80}} ATP to destroy one Ub5-DHFR molecule.","answer":"50–80","explanation":"Peth and colleagues combined the maximal degradation rate with ATP hydrolysis measured by a malachite green phosphate assay.","section":"evidence","topic":"numbers","sources":["evidence:atp-cost-ub5-dhfr","stat:ATP cost per tagged protein","fact:P9","ref:peth2013"],"tags":["atp","cost"],"difficulty":2,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:atp-cost-ub5-dhfr","machine":"proteasome","label":"ATP hydrolysed per ubiquitinated DHFR molecule degraded (Peth A 2013)","section":"evidence","anchor":"ev-atp-cost-ub5-dhfr"},{"source":"stat:ATP cost per tagged protein","machine":"proteasome","label":"Key number: ATP cost per tagged protein","section":"summary"},{"source":"fact:P9","machine":"proteasome","label":"ATP spent to destroy one tagged protein: 50–80 ATP","section":"story"},{"source":"ref:peth2013","machine":"proteasome","label":"Peth et al.","section":"sources","anchor":"ref-peth2013","href":"https://doi.org/10.1074/jbc.m113.482570"}]},{"id":"proteasome-folate-meaning","machine":"proteasome","kind":"qa","prompt":"Folic acid stabilises the DHFR fold. What happened to proteasome ATP use per minute and to degradation time when it was bound?","answer":"ATP use per minute stayed the same, but degradation slowed from 13 s to 23 s per molecule.","explanation":"So each molecule cost more, 90–140 ATP instead of 50–80: unfolding is a slow and costly step for a stable protein.","section":"evidence","topic":"numbers","sources":["evidence:folate-slows-degradation","step:The fold is pulled apart","ref:peth2013"],"tags":["unfolding","atp"],"difficulty":3,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:folate-slows-degradation","machine":"proteasome","label":"Effect of stabilising the DHFR fold with folic acid (Peth A 2013)","section":"evidence","anchor":"ev-folate-slows-degradation"},{"source":"step:The fold is pulled apart","machine":"proteasome","label":"Step: The fold is pulled apart","section":"mechanism"},{"source":"ref:peth2013","machine":"proteasome","label":"Peth et al.","section":"sources","anchor":"ref-peth2013","href":"https://doi.org/10.1074/jbc.m113.482570"}]},{"id":"proteasome-single-rpt-mutant","machine":"proteasome","kind":"qa","prompt":"Blocking ATP binding in one yeast proteasome ATPase (Rpt3, Rpt5 or Rpt6) cut basal ATP use by about 66%, not the 16% expected. What does that show?","answer":"The six ATPases run in an ordered cycle, so one stalled subunit holds up the others.","explanation":"If the subunits worked on their own, losing one of six would cost only about one sixth of the activity.","section":"evidence","topic":"cycle","sources":["evidence:rpt-single-mutant-atpase","step:Hand over hand","ref:peth2013"],"tags":["atpase","coordination"],"difficulty":3,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:rpt-single-mutant-atpase","machine":"proteasome","label":"Loss of basal ATPase activity when one Rpt subunit cannot bind ATP (Peth A 2013)","section":"evidence","anchor":"ev-rpt-single-mutant-atpase"},{"source":"step:Hand over hand","machine":"proteasome","label":"Step: Hand over hand","section":"mechanism"},{"source":"ref:peth2013","machine":"proteasome","label":"Peth et al.","section":"sources","anchor":"ref-peth2013","href":"https://doi.org/10.1074/jbc.m113.482570"}]},{"id":"proteasome-step-size","machine":"proteasome","kind":"qa","prompt":"In cryo-EM of working yeast 26S proteasomes, how far does the substrate move per ATP?","answer":"About 6 Å, read as two amino acids per ATP.","explanation":"De la Peña and colleagues saw the engaged pore-1 loop tyrosines move down by that distance between consecutive motor states.","section":"evidence","topic":"numbers","sources":["evidence:translocation-step-size","step:Hand over hand","ref:delapena2018"],"tags":["translocation","cryo-em"],"difficulty":2,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:translocation-step-size","machine":"proteasome","label":"Substrate translocation step per hydrolysed ATP (de la Peña AH 2018)","section":"evidence","anchor":"ev-translocation-step-size"},{"source":"step:Hand over hand","machine":"proteasome","label":"Step: Hand over hand","section":"mechanism"},{"source":"ref:delapena2018","machine":"proteasome","label":"de la Pena et al.","section":"sources","anchor":"ref-delapena2018","href":"https://doi.org/10.1126/science.aav0725"}]},{"id":"proteasome-mass-not-measured","machine":"proteasome","kind":"qa","prompt":"The human 26S proteasome is often given as 2.5 MDa, citing Dong et al. 2019. What is the problem with that source?","answer":"Dong et al. state the mass but do not measure it; a primary measurement still has to be found.","explanation":"Their paper is a cryo-EM structure study, and its own results are the seven structures, not a mass.","section":"evidence","topic":"debate","sources":["evidence:holoenzyme-mass","stat:Mass of the holoenzyme","ref:dong2019"],"tags":["mass","provenance"],"difficulty":2,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:holoenzyme-mass","machine":"proteasome","label":"Mass of the human 26S proteasome holoenzyme (Dong Y 2019)","section":"evidence","anchor":"ev-holoenzyme-mass"},{"source":"stat:Mass of the holoenzyme","machine":"proteasome","label":"Key number: Mass of the holoenzyme","section":"summary"},{"source":"ref:dong2019","machine":"proteasome","label":"Dong et al.","section":"sources","anchor":"ref-dong2019","href":"https://doi.org/10.1038/s41586-018-0736-4"}]}]}