T E C H N I C A L CO M M E N T
◥
LOCAL TRANSLATION
Comment on “Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling” David W. Reid1 and Christopher V. Nicchitta2* Jan et al. (Research Articles, 7 November 2014, p. 716) propose that ribosomes translating secretome messenger RNAs (mRNAs) traffic from the cytosol to the endoplasmic reticulum (ER) upon emergence of the signal peptide and return to the cytosol after termination. An accounting of controls demonstrates that mRNAs initiate translation on ER-bound ribosomes and that ribosomes are retained on the ER through many cycles of translation.
1
Program in Cardiovascular and Metabolic Disorders, DukeNUS Graduate Medical School, Singapore 169857, Singapore. 2 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA. *Corresponding author. E-mail: [email protected]
SCIENCE sciencemag.org
ribosomes are cotranslationally targeted from the cytosol to the ER after the signal sequence is translated, consistent with the signal hypothesis/ SRP pathway model. We were able to reproduce the authors’ primary observation using their data, where BirASsh1-labeled ribosomes were depleted on mRNAs encoding predicted signal sequences (10) until ~30 codons after the signal sequence (Fig. 1A). However, when we analyzed data from control experiments with a BirA-Ubc6 tail-anchor chimera (Ubc6TA), designed to be a reporter for general ER-associated translation, this ribosome depletion was not observed (Fig. 1B). Instead, the BirA-Ubc6TA reporter identified ER ribosomes translating secretome mRNAs throughout the open reading frame, including before the emergence of a signal sequence. This indicates that ribosomes were bound to the ER before signal sequence emergence, and thus that ribosome localization to the ER is not obligatorily coupled to SRP-signal sequence recruitment. To explain the paucity of ribosomes in the first ~30 codons
BirA-Ssh1
BirA-Ubc6 TA BirA labeling enrichment (Input / Pulldown)
BirA labeling enrichment (Input / Pulldown)
J
an et al. (1) report that ribosomes engaged in the translation of secretome mRNAs are recruited from the cytosol to the endoplasmic reticulum (ER) membrane early in translation and released back into the cytosol soon after completion of protein synthesis. Although these conclusions are consistent with the established signal hypothesis/signal recognition particle (SRP) pathway model (2, 3), recent work has demonstrated that ER-bound ribosomes translate both signal sequence– and cytosolic protein–encoding mRNAs and that ribosomes associate stably with the ER after termination (4–8). In an attempt to reconcile these discrepancies, we reanalyzed the authors’ data, including control data that were not integrated into their conclusions. We suggest that the data from Jan et al. rather demonstrate that ER-bound ribosomes initiate translation while remaining associated with the ER, translate both secretomeand cytosolic protein–encoding mRNAs, and stably associate with the ER through multiple cycles of translation. To examine ribosome trafficking and translation dynamics, Jan et al. developed proximityspecific ribosome profiling, where chimeras of resident ER membrane proteins and the biotin ligase BirA were used to selectively label ribosomes bearing BirA acceptor sequence–tagged ribosomal protein RPL16 (9). In one iteration, BirA-Ssh1, where Ssh1 is paralogous to the proteinconducting channel Sec61, was used to label translocon-proximal, ER-bound ribosomes. Jan et al. then determined the location of tagged ribosomes on mRNAs by ribosome profiling. The authors observed that few ribosomes were labeled by BirA-Ssh1 before the emergence of a signal sequence, leading them to conclude that
observed with the BirA-Ssh1 reporter, we suggest a scenario in which ER-bound ribosomes initiate translation from the ER-bound state and subsequently undergo signal sequence–dependent lateral recruitment to BirA-Ssh1 translocons, as an alternative to the cytosol to BirA-Ssh1 translocon ribosome trafficking model the authors propose. Because ER-bound ribosomes are well represented on secretome mRNAs before the emergence of the signal sequence, the data from Jan et al. demonstrate that cotranslational targeting to the ER is unlikely to be the sole, or even primary, mechanism by which mRNAs or ribosomes are localized to the ER If ribosomes are not recruited to the ER in a manner coupled to emergence of the signal sequence, how might the authors’ conclusions regarding ribosome exchange rates between the cytosol and ER be viewed? In Jan et al., ribosome exchange rates were determined by tracking the mRNA composition of BirA-Sec63–labeled ribosomes as a function of biotin labeling time. At short biotin pulse periods, BirA-Sec63–labeled ribosomes were highly enriched in secretome mRNAs. This enrichment dissipated within 7 min of labeling, at which point ribosomes translating cytosolic and secretome mRNAs were similarly labeled. These data led to the interpretation that labeled ribosomes that were previously translating ER proteins left the ER to initiate translation of cytosolic proteins, and so the composition of the tagged ribosome-associated mRNAs serves as a proxy for ribosome exchange. For this interpretation to be valid, ER-bound ribosomes must be largely devoted to the translation of secretome mRNAs. However, when analyzing the mRNA composition of labeled ribosomes during this time course, we found that the majority of tagged ribosomes were translating cytosolic proteins even after 1 min of biotin labeling, invalidating the assumption that ribosome subcellular location can be inferred from the composition of the associated mRNAs (Fig. 2A). This finding is consistent with several studies using diverse organisms and methods (Fig. 2B). The early enrichment for ribosomes translating ER proteins
Codons from 1st codon of signal
Codons from 1st codon of signal
Fig. 1. Ribosomes are bound to the ER before signal sequence emergence. The enrichment of ribosomes in the biotin-labeled pulldown relative to input was calculated at each codon for mRNAs that encode signal sequences. Plots were generated for (A) BirA-Ssh1 and (B) BirA-Ubc6TA. Shaded area represents mean T SD across all analyzed genes. Both analyses were performed on data from the 7-min biotin pulse without cycloheximide. 12 JUNE 2015 • VOL 348 ISSUE 6240
1217-a
Downloaded from www.sciencemag.org on June 11, 2015
R ES E A RC H
R ES E A RC H | T E C H N I C A L CO M M E N T
Encoding cytosolic proteins Encoding ER proteins
Percent of BirA-Sec63 labeled ribosomes
Percent of ER-bound transcriptome
Translating cytosolic proteins Translating ER proteins
Time labeling (min)
Species S. cerevisiae
REFERENCES AND NOTES
H. sapiens (HEK293) Detergent extraction
H. sapiens (K562) Equilibrium density centrifugation Diehn 2006
Method Equilibrium Fig. 2. The majority of ER-associated transdensity lation is devoted to cytosolic protein– centrifugation encoding mRNAs. (A) The fraction of Reference Diehn 2000 Reid 2012 BirA-Sec63–labeled ribosomes engaged in the translation of cytosolic or ER proteins was calculated at the indicated labeling times. (B) Stacked bar plot depicting the proportion of ER-associated mRNAs encoding cytosolic proteins. [Data are from (4, 5, 8)]
that the authors reported, instead of representing ribosome exchange, is likely due to a modest kinetic advantage for ribosomes in close proximity to translocon-associated BirA-Sec63. Although the half-life of a ribosome on the ER therefore remains unknown, the bulk of evi-
1217-a
12 JUNE 2015 • VOL 348 ISSUE 6240
localization is only weakly coupled to secretome protein targeting, and where the majority of ER-bound ribosomes translate cytosolic protein mRNAs.
dence that we have discussed above and elsewhere (11) indicates that it is substantially longer than a single cycle of translation. After additional analysis, the data from Jan et al. best support a model where ribosomes bind rather stably to the ER, where ribosome
1. C. H. Jan, C. C. Williams, J. S. Weissman, Science 346, 1257521 (2014). 2. P. Walter, A. E. Johnson, Annu. Rev. Cell Biol. 10, 87–119 (1994). 3. G. Blobel, B. Dobberstein, J. Cell Biol. 67, 835–851 (1975). 4. D. W. Reid, C. V. Nicchitta, J. Biol. Chem. 287, 5518–5527 (2012). 5. M. Diehn, M. B. Eisen, D. Botstein, P. O. Brown, Nat. Genet. 25, 58–62 (2000). 6. C. Zhou et al., Cell 159, 530–542 (2014). 7. S. B. Stephens, C. V. Nicchitta, Mol. Biol. Cell 19, 623–632 (2008). 8. M. Diehn, R. Bhattacharya, D. Botstein, P. O. Brown, PLOS Genet. 2, e11 (2006). 9. E. de Boer et al., Proc. Natl. Acad. Sci. U.S.A. 100, 7480–7485 (2003). 10. J. D. Bendtsen, H. Nielsen, G. von Heijne, S. Brunak, J. Mol. Biol. 340, 783–795 (2004). 11. R. M. Seiser, C. V. Nicchitta, J. Biol. Chem. 275, 33820–33827 (2000). AC KNOWLED GME NTS
The authors thank members of C.V.N. and Shirish Shenolikar’s laboratories. Work in C.V.N.’s laboratory is supported by a grant from the National Institute of General Medical Sciences of the U.S. National Institutes of Health (GM101533 to C.V.N.). D.W.R. is funded by a Translational Clinical Research Flagship Award entitled National Parkinson’s Disease Translational Clinical Research Programme from National Medical Research Council Singapore. 19 January 2015; accepted 8 April 2015 10.1126/science.aaa7257
sciencemag.org SCIENCE
Comment on ''Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling'' David W. Reid and Christopher V. Nicchitta Science 348, 1217 (2015); DOI: 10.1126/science.aaa7257
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Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2015 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on June 11, 2015
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◥
LOCAL TRANSLATION
Comment on “Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling” David W. Reid1 and Christopher V. Nicchitta2* Jan et al. (Research Articles, 7 November 2014, p. 716) propose that ribosomes translating secretome messenger RNAs (mRNAs) traffic from the cytosol to the endoplasmic reticulum (ER) upon emergence of the signal peptide and return to the cytosol after termination. An accounting of controls demonstrates that mRNAs initiate translation on ER-bound ribosomes and that ribosomes are retained on the ER through many cycles of translation.
1
Program in Cardiovascular and Metabolic Disorders, DukeNUS Graduate Medical School, Singapore 169857, Singapore. 2 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA. *Corresponding author. E-mail: [email protected]
SCIENCE sciencemag.org
ribosomes are cotranslationally targeted from the cytosol to the ER after the signal sequence is translated, consistent with the signal hypothesis/ SRP pathway model. We were able to reproduce the authors’ primary observation using their data, where BirASsh1-labeled ribosomes were depleted on mRNAs encoding predicted signal sequences (10) until ~30 codons after the signal sequence (Fig. 1A). However, when we analyzed data from control experiments with a BirA-Ubc6 tail-anchor chimera (Ubc6TA), designed to be a reporter for general ER-associated translation, this ribosome depletion was not observed (Fig. 1B). Instead, the BirA-Ubc6TA reporter identified ER ribosomes translating secretome mRNAs throughout the open reading frame, including before the emergence of a signal sequence. This indicates that ribosomes were bound to the ER before signal sequence emergence, and thus that ribosome localization to the ER is not obligatorily coupled to SRP-signal sequence recruitment. To explain the paucity of ribosomes in the first ~30 codons
BirA-Ssh1
BirA-Ubc6 TA BirA labeling enrichment (Input / Pulldown)
BirA labeling enrichment (Input / Pulldown)
J
an et al. (1) report that ribosomes engaged in the translation of secretome mRNAs are recruited from the cytosol to the endoplasmic reticulum (ER) membrane early in translation and released back into the cytosol soon after completion of protein synthesis. Although these conclusions are consistent with the established signal hypothesis/signal recognition particle (SRP) pathway model (2, 3), recent work has demonstrated that ER-bound ribosomes translate both signal sequence– and cytosolic protein–encoding mRNAs and that ribosomes associate stably with the ER after termination (4–8). In an attempt to reconcile these discrepancies, we reanalyzed the authors’ data, including control data that were not integrated into their conclusions. We suggest that the data from Jan et al. rather demonstrate that ER-bound ribosomes initiate translation while remaining associated with the ER, translate both secretomeand cytosolic protein–encoding mRNAs, and stably associate with the ER through multiple cycles of translation. To examine ribosome trafficking and translation dynamics, Jan et al. developed proximityspecific ribosome profiling, where chimeras of resident ER membrane proteins and the biotin ligase BirA were used to selectively label ribosomes bearing BirA acceptor sequence–tagged ribosomal protein RPL16 (9). In one iteration, BirA-Ssh1, where Ssh1 is paralogous to the proteinconducting channel Sec61, was used to label translocon-proximal, ER-bound ribosomes. Jan et al. then determined the location of tagged ribosomes on mRNAs by ribosome profiling. The authors observed that few ribosomes were labeled by BirA-Ssh1 before the emergence of a signal sequence, leading them to conclude that
observed with the BirA-Ssh1 reporter, we suggest a scenario in which ER-bound ribosomes initiate translation from the ER-bound state and subsequently undergo signal sequence–dependent lateral recruitment to BirA-Ssh1 translocons, as an alternative to the cytosol to BirA-Ssh1 translocon ribosome trafficking model the authors propose. Because ER-bound ribosomes are well represented on secretome mRNAs before the emergence of the signal sequence, the data from Jan et al. demonstrate that cotranslational targeting to the ER is unlikely to be the sole, or even primary, mechanism by which mRNAs or ribosomes are localized to the ER If ribosomes are not recruited to the ER in a manner coupled to emergence of the signal sequence, how might the authors’ conclusions regarding ribosome exchange rates between the cytosol and ER be viewed? In Jan et al., ribosome exchange rates were determined by tracking the mRNA composition of BirA-Sec63–labeled ribosomes as a function of biotin labeling time. At short biotin pulse periods, BirA-Sec63–labeled ribosomes were highly enriched in secretome mRNAs. This enrichment dissipated within 7 min of labeling, at which point ribosomes translating cytosolic and secretome mRNAs were similarly labeled. These data led to the interpretation that labeled ribosomes that were previously translating ER proteins left the ER to initiate translation of cytosolic proteins, and so the composition of the tagged ribosome-associated mRNAs serves as a proxy for ribosome exchange. For this interpretation to be valid, ER-bound ribosomes must be largely devoted to the translation of secretome mRNAs. However, when analyzing the mRNA composition of labeled ribosomes during this time course, we found that the majority of tagged ribosomes were translating cytosolic proteins even after 1 min of biotin labeling, invalidating the assumption that ribosome subcellular location can be inferred from the composition of the associated mRNAs (Fig. 2A). This finding is consistent with several studies using diverse organisms and methods (Fig. 2B). The early enrichment for ribosomes translating ER proteins
Codons from 1st codon of signal
Codons from 1st codon of signal
Fig. 1. Ribosomes are bound to the ER before signal sequence emergence. The enrichment of ribosomes in the biotin-labeled pulldown relative to input was calculated at each codon for mRNAs that encode signal sequences. Plots were generated for (A) BirA-Ssh1 and (B) BirA-Ubc6TA. Shaded area represents mean T SD across all analyzed genes. Both analyses were performed on data from the 7-min biotin pulse without cycloheximide. 12 JUNE 2015 • VOL 348 ISSUE 6240
1217-a
Downloaded from www.sciencemag.org on June 11, 2015
R ES E A RC H
R ES E A RC H | T E C H N I C A L CO M M E N T
Encoding cytosolic proteins Encoding ER proteins
Percent of BirA-Sec63 labeled ribosomes
Percent of ER-bound transcriptome
Translating cytosolic proteins Translating ER proteins
Time labeling (min)
Species S. cerevisiae
REFERENCES AND NOTES
H. sapiens (HEK293) Detergent extraction
H. sapiens (K562) Equilibrium density centrifugation Diehn 2006
Method Equilibrium Fig. 2. The majority of ER-associated transdensity lation is devoted to cytosolic protein– centrifugation encoding mRNAs. (A) The fraction of Reference Diehn 2000 Reid 2012 BirA-Sec63–labeled ribosomes engaged in the translation of cytosolic or ER proteins was calculated at the indicated labeling times. (B) Stacked bar plot depicting the proportion of ER-associated mRNAs encoding cytosolic proteins. [Data are from (4, 5, 8)]
that the authors reported, instead of representing ribosome exchange, is likely due to a modest kinetic advantage for ribosomes in close proximity to translocon-associated BirA-Sec63. Although the half-life of a ribosome on the ER therefore remains unknown, the bulk of evi-
1217-a
12 JUNE 2015 • VOL 348 ISSUE 6240
localization is only weakly coupled to secretome protein targeting, and where the majority of ER-bound ribosomes translate cytosolic protein mRNAs.
dence that we have discussed above and elsewhere (11) indicates that it is substantially longer than a single cycle of translation. After additional analysis, the data from Jan et al. best support a model where ribosomes bind rather stably to the ER, where ribosome
1. C. H. Jan, C. C. Williams, J. S. Weissman, Science 346, 1257521 (2014). 2. P. Walter, A. E. Johnson, Annu. Rev. Cell Biol. 10, 87–119 (1994). 3. G. Blobel, B. Dobberstein, J. Cell Biol. 67, 835–851 (1975). 4. D. W. Reid, C. V. Nicchitta, J. Biol. Chem. 287, 5518–5527 (2012). 5. M. Diehn, M. B. Eisen, D. Botstein, P. O. Brown, Nat. Genet. 25, 58–62 (2000). 6. C. Zhou et al., Cell 159, 530–542 (2014). 7. S. B. Stephens, C. V. Nicchitta, Mol. Biol. Cell 19, 623–632 (2008). 8. M. Diehn, R. Bhattacharya, D. Botstein, P. O. Brown, PLOS Genet. 2, e11 (2006). 9. E. de Boer et al., Proc. Natl. Acad. Sci. U.S.A. 100, 7480–7485 (2003). 10. J. D. Bendtsen, H. Nielsen, G. von Heijne, S. Brunak, J. Mol. Biol. 340, 783–795 (2004). 11. R. M. Seiser, C. V. Nicchitta, J. Biol. Chem. 275, 33820–33827 (2000). AC KNOWLED GME NTS
The authors thank members of C.V.N. and Shirish Shenolikar’s laboratories. Work in C.V.N.’s laboratory is supported by a grant from the National Institute of General Medical Sciences of the U.S. National Institutes of Health (GM101533 to C.V.N.). D.W.R. is funded by a Translational Clinical Research Flagship Award entitled National Parkinson’s Disease Translational Clinical Research Programme from National Medical Research Council Singapore. 19 January 2015; accepted 8 April 2015 10.1126/science.aaa7257
sciencemag.org SCIENCE
Comment on ''Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling'' David W. Reid and Christopher V. Nicchitta Science 348, 1217 (2015); DOI: 10.1126/science.aaa7257
This copy is for your personal, non-commercial use only.
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here.
The following resources related to this article are available online at www.sciencemag.org (this information is current as of June 11, 2015 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/348/6240/1217.1.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/348/6240/1217.1.full.html#related This article cites 11 articles, 6 of which can be accessed free: http://www.sciencemag.org/content/348/6240/1217.1.full.html#ref-list-1 This article has been cited by 1 articles hosted by HighWire Press; see: http://www.sciencemag.org/content/348/6240/1217.1.full.html#related-urls This article appears in the following subject collections: Cell Biology http://www.sciencemag.org/cgi/collection/cell_biol
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2015 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on June 11, 2015
Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here.
