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Response to Comment on “Principlesof ER cotranslational translocationrevealed by proximity-specificribosome profiling”Calvin H. Jan, Christopher C. Williams, Jonathan S. Weissman*
Reid and Nicchitta propose that most cellular translation is carried out by a noncyclingpool of endoplasmic reticulum (ER)–associated ribosomes. However, proximity-specificribosome profiling data place an upper bound of about 7 to 16% on the fraction of cytosolicprotein translation carried out by ribosomes accessible to ER-tethered biotin ligases.Moreover, yeast pulse-labeling experiments argue against there being a static population ofER-associated ribosomes.
Reid and Nicchitta have raised a fundamen-tal question in biology: Where in the celldoes translation occur? Inwhat representsa pronounced departure from the currenttextbook thinking (1), they argue that “the
ER is a primary site of general protein synthesis”(2) and that ribosomes stay stably associatedwith the endoplasmic reticulum (ER) throughmany rounds of translation. As they point out, ifthe ER were the primary site of translation ofboth secreted and cytosolic proteins, this wouldcomplicate the interpretation of our ribosome ex-change experiments.However, analysis of our datareveals that the large majority of cytosolic pro-teins are translated by a pool of ribosomes thatare inaccessible to ER-tethered biotin ligases(BirA), whereas a rapidly exchanging pool of ER-associated ribosomes strongly favors translationof secreted proteins.At the heart of this issue is the interpretation
of ourproximity-specific ribosomeprofiling enrich-ment data. The relative proportion of translationof nonsecretory proteins that is carried out byER-associated ribosomes (i.e., ribosomes that areaccessible to ER-tethered biotin ligases) can becalculated as b from the following equation re-lating secretory footprint representation in ourinput and pulldown libraries
qS,pulldown = qS,input/[qS,input + b(1 – qS,input)]
where qS is the fraction of footprints from ribo-somes translating secretory proteins (S) in the in-put (qS,input) or streptavidin pulldown (qS,pulldown)libraries, and b is the fraction of footprints from
nonsecretory proteins in streptavidin pulldownlibraries [x: (x ∉ S)] (3, 4). Importantly, b providesan upper bound on translation of nonsecretoryproteins at the ER because these footprints can befrom bona fide translation at the ER, nonspecificlabelingof cytosolic ribosomes, orbackgroundbind-ing. Indeed, it is likely that a low level of back-ground from the much larger pool of translationof cytosolic proteins contributes substantially to b.We calculated b for experiments labeling
ribosomes with the three different forms of ER-tethered BirA (BirA-Ssh1, Sec63-BirA, and BirA-Ubc6). This analysis revealed that at most 7 to16% of nonsecretory protein translation occurredon ribosomes that were accessible to ER-tetheredBirA (Table 1). Importantly, this is inconsistentwith the proposal by Reid and Nicchitta (5) thatBirA-Ubc6 biotinylates all ribosomes at the ERsurface (i.e., those translating both cytosolic andsecreted protein), whereas BirA-Ssh1 only bio-tinylates ribosomes translating secretory proteinsafter associating with the translocon, because theb’s for the two BirA fusions are comparable. Aninteresting open question is whether there is a“peri-ER” pool of ribosomes that remain in thevicinity of the ER but are not accessible to any ofthe ER-tethered BirA proteins.Reid and Nicchitta also present an analysis of
the codon-specific enrichmentof secretoryproteins.We disagree with this analysis on two grounds.The first is a conceptual one. They argue that it
was the observation that enrichment inBirA-Ssh1labeling occurs only after the emergence of thesignal sequence that led us “to conclude thatribosomes are cotranslationally targeted from thecytosol to the ER after the signal sequence istranslated.” This conflates the question of whichmRNAs are cotranslationally targetedwith atwhatpoint during translation targeting occurs. Infact, in our paper, we addressed the question ofcotranslational translocation [figure 3 in (3)] be-fore the introduction of position-specific analyses[figure 4 in (3)], which was motivated by a dis-tinct set of questions. Moreover, in the Jan et al.Research Article (3), we argue that our data arebest explained by a model in which, after aninitial round of targeting, mRNAs of secreted pro-tein remain tethered to the ER by downstreamtranslating ribosomes [figure 5C in (3)]. Thus, asubstantial fraction of the time, ribosomes willinitiate translation on messages for secretoryproteins that are tethered to the ER.The second disagreement is a technical one.
Despite claiming to “reproduce the authors’ pri-mary observation,” the metagene enrichmentspresented byReid andNicchitta both qualitative-ly (position dependence) and quantitatively (mag-nitude of enrichment) fail to recapitulate ourresults [compare Reid and Nicchitta’s figure 1 (5)with our Fig. 1 and with figure 4B in (3)]. It isnot clear to us how they arrived at their plots, be-cause insufficient details were provided and theypurport to analyze data sets (“7-min biotin pulsewithout cycloheximide” for BirA-Ssh1/BirA-Ubc6)that do not exist. In any case, it is clear that theyhave not correctly replicated our analysis.We havereproduced our results with three independentanalyses implemented by two individuals, and allbiotin ligase fusions showposition-specific enrich-ment that maximizes after the emergence of thesignal sequence/anchor (Fig. 1, code available onrequest).Reid and Nicchitta also argue that ribosomes
are stably associated with a static rough ER. Weobserved a rapid loss of the enrichment for se-creted proteins when biotinylation was allowedto proceed with ongoing translation (3). Reid andNicchitta argue that this is not due to exchange ofribosomes from the ER after translation termi-nation and new initiation. Rather, based on theirmodel that noncycling ER-associated ribosomestranslate mostly cytosolic proteins, they proposethat there are different kinetics of labeling fromER-localizedBirAof ribosomes translating secretedproteins and those translating cytosolic ones dueto “a modest kinetic advantage for ribosomes inclose proximity to translocon-associated BirA-Sec63.”However, this interpretation is ruled out by
RESEARCH
SCIENCE sciencemag.org 12 JUNE 2015 • VOL 348 ISSUE 6240 1217-b
Department of Cellular and Molecular Pharmacology, HowardHughes Medical Institute, California Institute for QuantitativeBiosciences, Center for RNA Systems Biology, University ofCalifornia–San Francisco, San Francisco, CA 94158, USA.*Corresponding author. E-mail: [email protected]
Table 1. Proportion of secretory footprints in data sets labeled for 7 min with cycloheximide.
Data set qS,input qS,pulldown b
BirA-Ubc6 0.14 0.62 0.10Sec63-BirA 0.13 0.47 0.16BirA-Ssh1 0.13 0.69 0.07Model expectation 0.13 <0.23 >0.5
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the persistent enrichment of secretome proteinsin the biotinylated pool through extended label-ing times when translation elongation, and thusribosome exchange, is inhibited with cyclohexi-mide (Fig. 2A) [see also figure 2A in (3)]. Thus, itis the act of translation termination and subse-quent initiation on a new message that allowsribosomes labeled on the ER surface to gain ef-ficient access to the pool of cytosolic messages.Additionally, the kinetic analysis presented by
Reid and Nicchitta in figure 2 in (5) is incorrect.The correct plot of the composition of footprintsin pulldown libraries during our pulse-labelingstudies reveals a robust enrichment for secretoryprotein that rapidly decays when labeling is al-lowed to proceed in the presence of ongoingtranslation (Fig. 2B). Importantly, although thereismarked enrichment after 1min of labeling, thisenrichment is substantially lower than when la-beling is carried out with cycloheximide (Fig. 2B).
Thus, even after 1 min (roughly the time it takesfor a single round of translation), there is alreadyconsiderable ribosome exchange that cannotbe accounted for by differences in BirA labelingkinetics.Finally, Reid and Nicchitta inaccurately char-
acterize our conclusions as being “consistentwith the established signal hypothesis/signal rec-ognition particle (SRP) pathway model.” Ourstudy was not meant to be a test, let alone a val-idation of the standard SRPmodel. Indeed, someof our findings clearly go beyond the canonicalSRP model, including the observations that SRP-independent proteins are cotranslationally tar-geted to the ER, that the duration of translationafter the emergence of a signal sequence/anchoris a major determinant of cotranslational trans-location, and that mRNAs can be targeted to theER even in the absence of SRP (3). Also, as dis-cussed above, we argue that after the initial re-
cruitment of a secretory protein message to theER, it can be retained there through tethering bymultiple ribosomes [figure 5C in (3)]. In sum-mary, our data demonstrate that, regardless ofthe initial targeting mechanism, the majorityof secretory proteins are translated at the ER,whereas the largemajority of cytosolic proteinsare not.
REFERENCES
1. B. Alberts, Molecular Biology of the Cell (Garland Science, Taylorand Francis Group, New York, ed. 6, 2015).
2. D. W. Reid, C. V. Nicchitta, Nat. Rev. Mol. Cell Biol. 16, 221–231(2015).
3. C. H. Jan, C. C. Williams, J. S. Weissman, Science 346, 1257521(2014).
4. We define S as the set of secretory proteins enriched above areceiver operator characteristic threshold that maximizedaccuracy for Sec63-BirA enrichment [figure S5 in (3)].
5. D. W. Reid, C. V. Nicchitta, Science 348, 1217 (2015).
14 February 2015; accepted 8 April 201510.1126/science.aaa8299
1217-b 12 JUNE 2015 • VOL 348 ISSUE 6240 sciencemag.org SCIENCE
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Fig. 1. Metagene analyses. (A) Metagene plot of log2 BirA-mVenus-Ssh1 enrichment per codon (mean T SD) as a function of ribosome position relative to thefirst codon of the first Phobius-predicted hydrophobic element for secretome proteins (excluding tail-anchored proteins). The heat map below representssingle-gene enrichments sorted by the number of codons before the first hydrophobic element. Cells were treated with 100 mg/ml cycloheximide and incubatedwith biotin for 2 min. (B) As in (A) for BirA-mVenus-Ubc6 enrichment. (C) As in (A) for Sec63-mVenus-BirA enrichment.
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Fig. 2. Kinetic analysis. (A) Bar graph showing thepercentage of footprints aligning to secretome pro-teins in streptavidin pulldown libraries from the indi-cated BirA-fusion protein after a 7-min incubation withbiotin. The dashed line shows the average percentage
of secretome footprints in the input libraries. (B) Plotted are the percentages of footprints in pulldown libraries that aligned to cotranslationally targetedsecretome genes (circles) or all other genes (triangles). Horizontal dashed lines indicate the percentage of footprints mapping to secretome (black) andnonsecretome (green) genes in whole-cell ribosome profiling libraries. The x axis indicates translation time after biotin addition.
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proximity-specific ribosome profiling''Response to Comment on ''Principles of ER cotranslational translocation revealed by
Calvin H. Jan, Christopher C. Williams and Jonathan S. Weissman
DOI: 10.1126/science.aaa8299 (6240), 1217.348Science
ARTICLE TOOLS http://science.sciencemag.org/content/348/6240/1217.2
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http://science.sciencemag.org/content/sci/346/6210/1257521.fullhttp://science.sciencemag.org/content/sci/348/6240/1217.1.full
REFERENCES
http://science.sciencemag.org/content/348/6240/1217.2#BIBLThis article cites 3 articles, 2 of which you can access for free
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