PodcastCell Biology

Science Signaling Podcast: 8 November 2011

Science Signaling  08 Nov 2011:
Vol. 4, Issue 198, pp. pc24
DOI: 10.1126/scisignal.2002580


This Podcast features a conversation with the senior author of a Report published in the 4 November 2011 issue of Science. Signaling through mTORC1 (the mammalian target of rapamycin complex 1) promotes cell growth by stimulating protein synthesis. The activity of mTORC1 is regulated by cellular nutrient and energy status as well as growth factors. The presence of amino acids stimulates the translocation of mTORC1 to the surface of the lysosome, where it is activated. Zoncu and colleagues report that the vacuolar ATPase is required for amino acids to activate mTORC1. Senior author David Sabatini discusses these findings.

(Length: 11 min; file size 6.2 MB; file format: mp3; location: http://podcasts.aaas.org/science_signaling/ScienceSignaling_111108.mp3)

Technical Details

Length: 11 min

File size: 6.2 MB

File Format: mp3

RSS Feed: http://stke.sciencemag.org/rss/podcast.xml

Listen to Podcast: http://podcasts.aaas.org/science_signaling/ScienceSignaling_111108.mp3

Educational Details

Learning Resource Type: Audio

Context: High school upper division 11-12, undergraduate lower division 13-14, undergraduate upper division 15-16, graduate, professional, general public and informal education

Intended Users: Teacher, learner

Intended Educational Use: Learn, teach

Discipline: Cell biology, molecular biology

Keywords: Science Signaling, amino acid sensing, GTPase, lysosome, mammalian target of rapamycin complex 1, mTORC1, proton pump, Rag, Ragulator, Rheb, vacuolar ATPase, V-ATPase


Host – Annalisa VanHookWelcome to the Science Signaling Podcast for November 8th, 2011. I’m Annalisa VanHook, and today I’m speaking with David Sabatini about a study that identifies a requirement for the vacuolar ATPase in activating signaling through mTORC1 (1). mTORC1—the mammalian target of rapamycin complex 1—promotes cell growth by stimulating protein synthesis. The activity of mTORC1 is regulated by cellular nutrient and energy status, as well as by growth factors. The presence of amino acids stimulates translocation of mTORC1 to the lysosomal surface where it’s activated. In a new study published in the current issue of Science, Zoncu and colleagues report that the vacuolar ATPase is required for amino acids to activate mTORC1. Sabatini spoke to me from the Massachusetts Institute of Technology.

Interviewer – Annalisa VanHookWelcome, Dr. Sabatini.

Interviewee – David SabatiniThank you.

Interviewer – Annalisa VanHookmTORC1 activity is stimulated by the presence of amino acids, which indicates that the cell has enough of the raw materials that it needs to make new proteins and lipids and ATP that will enable the cell to grow. How do amino acids stimulate mTORC1 activity?

Interviewee – David SabatiniYeah, so this is a question that we’ve been interested in for a long time and I think is one of the bigger holes actually in the entire mTOR field. It turns out the answer is quite complicated and quite distinct than what we might expect. And so, I think what we’ve found over the last few years is that what happens is [that] the amino acids cause mTORC1 to translocate to the surface of the lysosome, and there it can interact with an activator called Rheb, which is a small GTPase. And Rheb turns on the kinase activity of mTORC1. And so, really, what amino acids do is allow mTORC1 to find its activator, which, again, we think lives on the surface of the lysosome. And so, there appears to be a whole molecular machinery to do amino acid sensing and to cause this translocation, and we’ve been trying to work out what that machinery actually is.

Interviewer – Annalisa VanHookIt’s been unclear where the amino acid signaling that initiates mTORC1 activation takes place in the cell. Why is the location of that activation important?

Interviewee – David SabatiniWell, I think for a couple of reasons. One is that we do think there is this spatial separation between mTORC1 and its activator, Rheb, and that you need to get them together so that activation occurs. The question as to why this seems to be happening on the lysosomal surface—at least our evidence suggests that’s the case—is an interesting one, and we don’t fully understand the “why” aspect of this. Our current hypothesis is that the amino acid content of lysosomes is somehow important to the cell in telling it whether to grow or not. You might imagine that perhaps lysosomes are storing amino acids or that, for some reason, that amino acids being inside the lysosomes are important to the cell to making the decision to grow. And so, our current operating model is that mTORC1 really senses lysosomal amino acids, and to some extent, the idea that lysosomes have a key role in amino acid metabolism is really, you know, it’s a new one, but in retrospect maybe is not so shocking because lysosomes are where autophagosomes fuse and autophagy takes place and the amino acids are liberated. In yeast, the lysosomal equivalent, which is called the vacuole, stores amino acids. So, if you had to put a major growth sensor that cares about amino acids, maybe on the surface of a lysosome is not such a bad place to do it.

Host – Annalisa VanHookGiven the observation that the GTPases that activate mTORC1 are localized to the surface of the lysosome, Sabatini’s group investigated the lysosome as a potential location for the amino acid–induced signaling that leads to mTORC1 activation. To do that, they knocked down the expression of genes that are required for formation and function of the lysosome and looked for any that had an effect on mTORC1 activity. They identified the vacuolar ATPase as required for mTORC1 activation.

Interviewee – David SabatiniWe first started investigating the lysosome because, when we started working out the machinery that causes this translocation, we identified another class of small GTPases called the Rag GTPases and then a complex that we ended up calling the Ragulator that regulated the Rags. And all of that seemed to be localized on the lysosomal surface. And so, you know, why is that? We didn’t think this was just by chance that the lysosome was picked. And so, we said, “Okay, well, maybe it’s not only localizations of the lysosome that matters, but perhaps actually the function of lysosome matters.” And so, what we started to do is take many sort of classic genes that are associated with lysosomal function—and most are at the lysosome, the gene products are—and to eliminate them by RNAi and to ask, “Does anything happen to the mTORC1 pathway?” And many of them didn’t. The ones that did were components of a large protein complex called the vacuolar ATPase—or the V-ATPase for short. This is sort of a classic lysosomal enzyme complex. It has a historical name, vacuolar ATPase, but it’s also found in the lysosome, as well as in late endosomes—in fact, in other parts of the endomembrane system. And this is a large protein complex that, what it does is, it uses the energy in ATP to pump protons into the lysosome and to acidify it. And so, we found using both genetic means—RNAi—as well as pharmacological means, that the V-ATPase was necessary for mTORC1 to be activated in response to amino acids.

Interviewer – Annalisa VanHookHow does that proton pumping activity—the acidification of the lysosome—promote mTORC1 activation?

Interviewee – David SabatiniRight. And I think that’s an important question, and the truthful answer is [that] we’re not sure yet. The V-ATPase is very large—it has at least 16 subunits. And so, the possibility exists that it has other functions besides the proton pumping function that may be important in amino acid sensing. And the molecules that we use to inhibit the V-ATPase really block all potential function of the V-ATPase because it prevents it from using ATP. And so, we think the V-ATPase is somewhere between amino acids and the regulation of these Rag GTPases that I mentioned to you before are key for this translocation event. But exactly where—is it the amino acid sensor, is it part of the relay pathway? We don’t know where exactly it is, but that it’s absolutely necessary, and that it interacts itself with components of this Rag GTPase machinery is true. But we don’t know where exactly in the pathway it stands. It’s certainly upstream of the Rags but downstream of amino acids.

Interviewer – Annalisa VanHookSo, the activity of the vacuolar ATPase is required to acidify the lysosome, which is required for that organelle to carry out its function of breaking down unneeded or unused cellular components to liberate the amino acids so that they can be reused. Do you think that the function of the vacuolar ATPase is just required to allow the lysosome create the free amino acids that can then activate mTORC1?

Interviewee – David SabatiniYeah. And at first, when we first found this, that was what we imagined would be the case—is that the V-ATPase is required for proteins to be broken down into amino acids, as well as for amino acids to be imported into lysosomes, because much of the activity of the lysosome depends on that proton gradient or on the acid environment that the protons generate. But what we did is we did is—Roberto, who led this work—what he did is, he ended up using amino acids that were derivatized in a way that they could get into the lysosome even if there was no proton gradient. And what we found is that the V-ATPase is still required even if you get amino acids into the lysosome in the absence of its activity. And so, it may be important for bringing amino acids in or for proteins to be degraded—it almost certainly is. But, it seems to have another function, as well. And I think we’ve shown that quite carefully—that the function of the V-ATPase is more than simply having protein breakdown or amino acid entry into the lysosomes.

Interviewer – Annalisa VanHookHas there been any sort of analogous functions for the vacuolar ATPase required for something besides just making the proton gradient in other systems, like in yeast?

Interviewee – David SabatiniWell, there have been some data suggesting that the V-ATPase has a role in glucose sensing in yeast—there’s proteins that bind to it in a glucose-sensitive fashion, it appears. There’s also some data suggesting that the V-ATPase plays a role involved in actually acid sensing, in pH sensing inside of the lysosome. So, I don’t think it’s going to be shocking if we find more and more functions of the V-ATPase. At the end of the day, it’s a very big protein complex—parts seem to come on and off; its assembly is regulated. So it’s a complicated machine, and so the fact that one might find more and more functions for it, you know, you might expect that.

Interviewer – Annalisa VanHookDo any of these components of the V-ATPase physically interact with the regulatory apparatus that activates mTORC1?

Interviewee – David SabatiniSo, mTORC1 is translocated to the lysosomal surface, and really the binding site seems to be these proteins that we call the Rag GTPases. They, in turn, bind to a large complex called the Ragulator, which is a multiprotein complex. That Ragulator binds to the V-ATPase in an amino acid–sensitive way—or at least the interaction between them is regulated by amino acids. So there is a direct physical interaction. And, in fact, when we had done many different protein purification effort[s], we always found V-ATPase subunits in our purifications, but because we knew it was an abundant protein in the lysosome, we pretty much ignored them. And, at one point, we said, “Whoa, why are we ignoring this sort of major component of our purifications?” It was good specificity in the purifications, and we were just sort of biased against it because we knew that it was a fairly abundant protein complex.

Interviewer – Annalisa VanHookThank you, Dr. Sabatini, for taking the time to speak with me.

Interviewee – David SabatiniWell, thank you.

Host – Annalisa VanHookThat was David Sabatini, senior author of a Report published in the November 4th issue of Science. That paper, by Zoncu and colleagues, is titled “mTORC1 Senses Lysosomal Amino Acids Through an Inside-Out Mechanism That Requires the Vacuolar H+-ATPase” (1).


And that wraps up this Science Signaling Podcast. If you have any questions or suggestions, please write to us at sciencesignalingeditors{at}aaas.org. This show is a production of Science Signaling and of AAAS—Advancing Science, Serving Society. I'm Annalisa VanHook, and on behalf of Science Signaling and its publisher, the American Association for the Advancement of Science, thanks for listening.


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