How do pulse chase experiments work

We here reveal the existence of an ubiquitin-dependent folding machinery, which operates on the cytosolic side of the ER membrane Figure 4C. More specifically we show that following synthesis, LRP6 undergoes ubiquitination on Lys, most likely of a specific form. In conceptual analogy to the binding of newly synthesized mono-glucosylated proteins to calnexin in the ER lumen Lamriben et al. Or, LRP6 can undergo a new cycle of Lysspecific ubiquitination and chaperone binding. In the ER, LRP6 can thus undergo two types of ubiquitination events, which operate with different kinetics: 1 specific ubiquitination on Lys which promotes folding, 2 the slower polyubiquitination on other lysines which promotes ERAD.

These two types of ubiquitination events are reminiscent of de-glucosidation and de-mannosidation of glycoproteins in the ER lumen, which also occur with different kinetics. De-glucosidation promotes folding of glycosylated protein, but if folding is too lengthy or fails, de-mannosidation takes place and targets the protein to ERAD. While the here described ubiquitin-dependent folding system was identified for LRP6, it likely also operates on other membrane proteins. Future studies are required to establish the generality of this ubiquitin-dependent folding system, determine whether it has a built-in quality control, and identify the missing components such as the folding-promoting ubiquitin ligase, the putative ubiquitin-binding chaperone and the degradation-targeting ligase Figure 4C.

All these enzymes could be exploited to control Wnt signaling in the context of disease. Hela and RPE1 cells were used in this study. These cells are not on the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee.

The Knockout clones were detected by western blot Figure 1—figure supplement 1B. LRP6 encoding plasmids as been previously described Abrami et al. For the dual Luciferase assay, plasmids and reagents were from Promega. Supernatants were incubated overnight with Sepharose beads. In case of non-coupled Sepharose G beads, supernatants were subjected to preclearing with the beads prior to the actual immunoprecipitation reaction.

Cell surface protein biotinylation was performed as described Abrami et al. Briefly, silenced cells were treated 30 min with 0. The lysate was immunoprecipitated with streptavidin-coated sepharose beads Sigma, S The dual luciferase assays were performed with plasmids and reagents from Promega.

HEK cells were transfected with 0. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Ubiquitin-dependent folding of the Wnt signaling coreceptor LRP6" for consideration by eLife. Your article has been favorably evaluated by Ivan Dikic Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors.

The reviewers have opted to remain anonymous. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. There is a clear consensus between the reviewers that this paper is interesting and describes a significant new form of quality control of membrane protein synthesis and maturation.

Newly synthesized LRP6 is stabilized by ubiquitination of K, allowing folding and palmitoylation. Subsequent removal of ubiquitin is necessary for forward trafficking to the Golgi apparatus.

Despite the general positivity among reviewers, there are some issues that need to be resolved before it can be accepted. Investigating the glycosylation in this way should also address the above question of whether LRP6 is trafficked conventionally through the Golgi. This is a major mechanistic issue and should be explored a little further. The consequences on Wnt signaling of overexpression of a catalytically dead form of USP19 would be a simple experiment that might separate these functions, allowing the possibility of this dual function to be addressed.

K ubiquitination is both a stabilizing process for the newly synthesized LRP6 — allowing a chaperone to bind and support folding. This appears to contradict the idea that ubiquitination is stabilizing.

The resolution of this paradox is probably the timing of two events: first stabilization by ubiquitin addition, then ubiquitin removal for ER exit — but this is not well described, nor is there much data to support it strongly.

Do they think that the palmitoylation, which is discussed but not well integrated into the model, is a secondary stabilizing factor that prevents destabilization of mature LRP6 after the ubiquitin is removed? And is there anything to be said about the relationship between K ubiquitination and poly-ubiquitination that promotes ERAD? Please clarify the model generally. It would also be of interest to see how overexpressing the USP19 catalytic mutant would affect cell surface expression of LRP6.

We now mention in the Introduction third paragraph that upon exit from the ER, LRP6 is transported through the Golgi to the plasma membrane as indicated by the lack of EndoH sensitivity of the mature protein Abrami et al. Thus degradation by the Bafilomycin sensitivity pathway requires ER exit. We show in Figure 3C that over- expression of catalytically inactive USP19 has no protective role towards degradation.

Our initial interpretation was limited to the conclusion that the catalytic activity is required for LRP6 rescue, but the complete lack of effect also rules out that USP19 acts as the chaperone since otherwise a partial rescue would have been observed. The latter does not have time to take place if the protein folds and exits the ER. We also mention that, given the proximity of the palmitoylation sites to Lys, palmitoylation might prevent re-ubiquitination on Lys, thus preventing re- entry into the cycles of ubiquitination-deubiquitination.

Finally, we mention that the two types of ubiquitination events are reminiscent of de-glucosidation and de-mannosidation of glycoproteins in the ER lumen, which also occur with different kinetics. De-glucosidation promotes folding but if this is too lengthy de-mannosidation takes place and targets the protein to ERAD.

We thank the reviewers for raising this point. We have now removed this figure, which indeed lacked the appropriate controls. Almost all experiments in the paper were performed by metabolic pulse-chase because, as we mentioned already in the original text, western blot analysis of the steady state population does not allow the analysis of the effect of USP19 by protein overexpression, due to the very high stability of a subpopulation of LRP6, WT and mutants, that end up constituting the steady state population monitored by western blot.

Consistent with this, as we should have realized, surface biotinylation, which is revealed by western blotting, with the requested controls, does not, and cannot, reveal differences of surface LRP6 upon overexpression. Thus, while the Wnt signaling assay, which is more quantitative, indicates that cells overexpressing LRP6, Mesd and USP19 undergo higher Wnt signaling than cells overexpressing only LRP6 and Mesd Figure 4A in cells that have endogenous levels of the 3 proteins , this cannot be seen by surface biotinylation.

You'll find the correct answer below During a pulse-chase experiment, photographic emulsions were prepared at different times during the chase, and radioactive spots were detected at the following times and locations: 5 minutes: rough ER; 10 minutes: Golgi apparatus; 40 minutes: endosomes; 70 minutes: lysosomes; minutes: lysosomes.

The proteins traveled from lysosomes to endosomes. The proteins did not travel through the Golgi apparatus. The proteins were secreted. The Correct Answer is The final destination of the proteins was the lysosome. Category: mastering bio. Related Blogs Who is responsible for managing the progress of work during a Sprint? This fact led to a dilemma regarding how the semiconservative model would work for a DNA molecule.

Reiji Okazaki was a brilliant experimenter who took on this problem. As an aside, Okazaki was born near Hiroshima, Japan, in He was a teenager there at the time of the explosion of the first of two nuclear bombs that the US dropped at the end of World War II. Reiji's scientific career was cut short by his untimely death from cancer in at the age of 44, perhaps related to his exposure to the fallout of that blast.

Okazaki reasoned that there were three possibilities for replicating a double-stranded DNA molecule, shown in Figure The continuous model is impossible, based upon the nature of DNA polymerases replication in only one direction. Therefore he needed to demonstrate that one of the other two models was actually taking place. Any discontinuous synthesis requires that there be, at least transiently, small pieces of DNA in a replicating structure.

Okazaki decided to look for these small pieces. He employed the ultracentrifuge to do this. This time, the separation is based upon size, so that he could see these smaller pieces and also follow what happens to them during replication. This time, notice that the relative motion of the molecules the S value is directly related to the size M.