WEBVTT 00:00:17.040 --> 00:00:21.160 BARBARA IMPERIALI: I always like to just remind you that 00:00:21.160 --> 00:00:22.620 the sixth -- 00:00:22.620 --> 00:00:24.810 it's kind of an assignment, but the numbers-- 00:00:24.810 --> 00:00:28.260 we're going to do this news brief project, where 00:00:28.260 --> 00:00:30.930 it's a teamwork project if you choose. 00:00:30.930 --> 00:00:35.030 If you take a look at the piece that you have in your hands 00:00:35.030 --> 00:00:38.190 now, it asks you for a little bit of information 00:00:38.190 --> 00:00:40.710 on that, who you're going to be working with, 00:00:40.710 --> 00:00:42.720 if you choose to work with someone. 00:00:42.720 --> 00:00:44.110 Or you can work on your own. 00:00:44.110 --> 00:00:45.137 That's fine. 00:00:45.137 --> 00:00:46.970 And we're looking to get a news brief that's 00:00:46.970 --> 00:00:51.510 of significance to research going on in the life sciences. 00:00:51.510 --> 00:00:53.580 And I've given you-- there are a couple of links 00:00:53.580 --> 00:00:56.880 in the sidebar of the website, so good places where you 00:00:56.880 --> 00:00:59.040 can find interesting material. 00:00:59.040 --> 00:01:02.150 What I'm super interested in for you, 00:01:02.150 --> 00:01:05.930 as a group where many of you are in the engineering fields, 00:01:05.930 --> 00:01:08.760 is to find something really cool at the interface 00:01:08.760 --> 00:01:11.240 between the life sciences and engineering, 00:01:11.240 --> 00:01:15.240 where engineering has a huge impact on the life sciences. 00:01:15.240 --> 00:01:16.810 You have alternatives. 00:01:16.810 --> 00:01:19.500 You can download the coordinates of a protein 00:01:19.500 --> 00:01:22.470 and print it on a 3D printer and give us 00:01:22.470 --> 00:01:25.360 a summary of what the protein is, what it does, 00:01:25.360 --> 00:01:27.420 and submit your 3D print. 00:01:27.420 --> 00:01:28.920 I'll give it back to you afterwards, 00:01:28.920 --> 00:01:30.240 once we've had a look at it. 00:01:30.240 --> 00:01:33.180 But actually submit the 3D print. 00:01:33.180 --> 00:01:35.160 And then the other opportunity is-- 00:01:35.160 --> 00:01:37.290 I think you'll remember back to when 00:01:37.290 --> 00:01:40.600 we were talking about molecular biology of the cell. 00:01:40.600 --> 00:01:43.080 I did kind of a clunky demo at the front 00:01:43.080 --> 00:01:45.540 of the class, nothing like Professor Martin's demos 00:01:45.540 --> 00:01:46.440 at all. 00:01:46.440 --> 00:01:49.230 This was me with the ethernet cables showing you 00:01:49.230 --> 00:01:51.270 what topoisomerase did. 00:01:51.270 --> 00:01:53.250 But in my demo, I didn't show you 00:01:53.250 --> 00:01:56.850 how topo also cuts a strand of DNA, 00:01:56.850 --> 00:01:59.940 holds it while the supercoiling unwinds, 00:01:59.940 --> 00:02:01.490 and then stitches it together. 00:02:01.490 --> 00:02:03.007 So I thought some of the engineers 00:02:03.007 --> 00:02:04.590 might be able to come up was something 00:02:04.590 --> 00:02:07.210 that was really better than that for me to use, 00:02:07.210 --> 00:02:09.310 for us to use, next year in class. 00:02:09.310 --> 00:02:13.680 So I'm really laying down the challenge there. 00:02:13.680 --> 00:02:16.470 So I always like things in the news. 00:02:16.470 --> 00:02:18.120 I thought this was kind of interesting 00:02:18.120 --> 00:02:21.630 that the first vertebrates evolved in shallow waters. 00:02:21.630 --> 00:02:25.470 I thought those were really cool first vertebrates. 00:02:25.470 --> 00:02:27.900 I'd love to get one of them in a fish tank and keep it. 00:02:27.900 --> 00:02:32.270 But anyway, that's that. 00:02:32.270 --> 00:02:35.730 It's truly amazing what you can see in the science 00:02:35.730 --> 00:02:37.710 reports, news briefs. 00:02:37.710 --> 00:02:39.660 I look at them whenever they come in. 00:02:39.660 --> 00:02:41.940 I get the posts every two or three days. 00:02:41.940 --> 00:02:43.920 And I'm kind of pleased to see that there's 00:02:43.920 --> 00:02:46.890 a lot of things that are in those news briefs 00:02:46.890 --> 00:02:49.260 that I feel that we're enabling you to read 00:02:49.260 --> 00:02:51.570 with some appreciation because of what 00:02:51.570 --> 00:02:53.440 we're covering in the class. 00:02:53.440 --> 00:02:56.310 So what we're doing now is we're really taking a leap forward 00:02:56.310 --> 00:03:00.000 here into cells and organisms, with respect 00:03:00.000 --> 00:03:10.140 to understanding how structure and function 00:03:10.140 --> 00:03:14.130 of individual macromolecules, proteins, nucleic acids, 00:03:14.130 --> 00:03:18.420 sugars, determine life, determine the dynamics of life 00:03:18.420 --> 00:03:22.530 that are necessary for an organism to really go 00:03:22.530 --> 00:03:25.920 through a life cycle, divide, have cells divide, 00:03:25.920 --> 00:03:27.660 go forward, have cells move. 00:03:27.660 --> 00:03:29.070 So what we're going to be talking 00:03:29.070 --> 00:03:32.400 about in the next lectures, which is section 6, 00:03:32.400 --> 00:03:34.680 is cellular trafficking and signaling. 00:03:34.680 --> 00:03:38.370 And so for the first lecture, which is 19 that we're on now-- 00:03:38.370 --> 00:03:40.680 so we're past the midway mark-- 00:03:40.680 --> 00:03:42.600 I'm going to be talking about trafficking. 00:03:42.600 --> 00:03:44.730 And that is how, within a cell, things 00:03:44.730 --> 00:03:49.260 get to where they need to be, or they get exported from a cell. 00:03:49.260 --> 00:03:51.690 Because all of the actions of a cell-- 00:03:51.690 --> 00:03:54.180 I really like thinking about the cell 00:03:54.180 --> 00:03:56.910 as a circuit board, where there's a receiver that 00:03:56.910 --> 00:03:58.530 gets information. 00:03:58.530 --> 00:04:03.000 And then the complex circuitry determines what outcome 00:04:03.000 --> 00:04:04.740 you get at the end of the day. 00:04:04.740 --> 00:04:07.680 So many of the proteins that we've talked about 00:04:07.680 --> 00:04:12.300 need to be in specific places for the cell to function. 00:04:12.300 --> 00:04:15.820 We have to have DNA polymerase in the nucleus. 00:04:15.820 --> 00:04:18.730 It's not going to be useful in the cytoplasm. 00:04:18.730 --> 00:04:24.270 We have to have a transcription factor that helps transcription 00:04:24.270 --> 00:04:27.540 go to the nucleus at the right time for transcription 00:04:27.540 --> 00:04:28.530 to occur. 00:04:28.530 --> 00:04:30.330 But we don't want it there all the time, 00:04:30.330 --> 00:04:32.205 because otherwise you'd have the light switch 00:04:32.205 --> 00:04:33.600 on the entire time. 00:04:33.600 --> 00:04:34.620 That wouldn't be useful. 00:04:34.620 --> 00:04:39.060 So we need to regulate where certain macromolecules are. 00:04:39.060 --> 00:04:42.570 We need to have the receivers on the surface of the cell 00:04:42.570 --> 00:04:45.010 to receive signals from outside. 00:04:45.010 --> 00:04:48.760 This is not just pertinent for multicellular organisms. 00:04:48.760 --> 00:04:51.420 It's pertinent for unicellular organisms, 00:04:51.420 --> 00:04:53.700 for them to sense their environment, 00:04:53.700 --> 00:04:55.640 know what's going on around them. 00:04:55.640 --> 00:04:57.690 Is the salt concentration changing? 00:04:57.690 --> 00:04:58.830 Is it getting very hot? 00:04:58.830 --> 00:05:00.240 Is it getting cold? 00:05:00.240 --> 00:05:02.010 Is there enough oxygen? 00:05:02.010 --> 00:05:05.910 Even unicellular organisms need to receive signals and respond 00:05:05.910 --> 00:05:06.780 to them. 00:05:06.780 --> 00:05:10.950 Multicellular organisms are way more complicated. 00:05:10.950 --> 00:05:14.010 Because you need to establish organs 00:05:14.010 --> 00:05:16.770 and different parts of a multicellular organism 00:05:16.770 --> 00:05:18.630 that have specialized function. 00:05:18.630 --> 00:05:21.540 So trafficking really is about what 00:05:21.540 --> 00:05:26.220 happens after you've made a replicated DNA in the nucleus, 00:05:26.220 --> 00:05:30.660 transcribed it, made a mature messenger that goes out 00:05:30.660 --> 00:05:32.520 to the cytoplasm in most cases. 00:05:32.520 --> 00:05:35.070 We'll talk about the exceptions to that case. 00:05:35.070 --> 00:05:38.250 And then in the cytoplasm, when proteins are expressed, 00:05:38.250 --> 00:05:40.800 all the different things that happen that 00:05:40.800 --> 00:05:44.070 guarantee that the protein gets to a proper destination 00:05:44.070 --> 00:05:45.270 for function. 00:05:45.270 --> 00:05:47.400 And some of those are quite complicated. 00:05:47.400 --> 00:05:49.620 Because remember, if I'm going to park 00:05:49.620 --> 00:05:52.470 a receiver in the cellular membrane 00:05:52.470 --> 00:05:55.320 with the signals being captured from outside, 00:05:55.320 --> 00:05:56.910 I've got to get from the cytoplasm 00:05:56.910 --> 00:06:00.780 out there in a reliable way. 00:06:00.780 --> 00:06:04.470 In lectures 20 and 21, I'll talk to you 00:06:04.470 --> 00:06:08.610 about cellular signaling with a focus on mammalian cells 00:06:08.610 --> 00:06:10.890 and the sorts of signaling processes 00:06:10.890 --> 00:06:15.600 that may go awry in cells, for example, proliferating cells. 00:06:15.600 --> 00:06:17.820 And then Professor Martin will really 00:06:17.820 --> 00:06:22.620 focus in on neuronal cells, optogenetics in lecture 22. 00:06:22.620 --> 00:06:26.280 So this bundle really allows you to call in the things 00:06:26.280 --> 00:06:28.740 that you've learned until now and apply them 00:06:28.740 --> 00:06:32.280 into much more intriguing and complex situations. 00:06:32.280 --> 00:06:34.380 So here's a wonderful, sort of silly drawing 00:06:34.380 --> 00:06:36.390 of a triangular cell. 00:06:36.390 --> 00:06:41.490 There's always a joke in cell biologists, 00:06:41.490 --> 00:06:45.300 when they're trying to talk to mathematicians 00:06:45.300 --> 00:06:48.000 and mathematicians want to simplify everything. 00:06:48.000 --> 00:06:50.040 And so everything gets-- imagine a cell, 00:06:50.040 --> 00:06:52.470 and there's this box shows up on a screen. 00:06:52.470 --> 00:06:54.540 Well, we all know that cells aren't 00:06:54.540 --> 00:06:56.310 triangular or box-shaped. 00:06:56.310 --> 00:06:59.310 But nevertheless, I thought this one was particularly cool. 00:06:59.310 --> 00:07:02.730 And so trafficking, the process of trafficking, 00:07:02.730 --> 00:07:07.770 is really all about, where is the information encoded 00:07:07.770 --> 00:07:11.670 into the protein that ensures that the protein is where 00:07:11.670 --> 00:07:16.920 it needs to be for the dynamics that we observe 00:07:16.920 --> 00:07:19.930 in living system? 00:07:19.930 --> 00:07:22.020 We've talked a lot about static things. 00:07:22.020 --> 00:07:23.268 We make the protein. 00:07:23.268 --> 00:07:24.060 Here's the protein. 00:07:24.060 --> 00:07:25.410 The protein folds. 00:07:25.410 --> 00:07:28.470 We've talked a lot about things that are kind of fixed 00:07:28.470 --> 00:07:29.590 in time and space. 00:07:29.590 --> 00:07:31.380 But what we want to do is understand 00:07:31.380 --> 00:07:35.760 what makes a cell programmed to undergo a new function. 00:07:35.760 --> 00:07:38.580 For example, something as simple as cell division, 00:07:38.580 --> 00:07:43.800 we have to orchestrate a huge variety of activities in order 00:07:43.800 --> 00:07:46.770 for the cell division process to start to occur. 00:07:46.770 --> 00:07:50.190 Something as really simple a cell mobility, think about, 00:07:50.190 --> 00:07:52.230 how do cells move? 00:07:52.230 --> 00:07:54.540 They're not moving all the time, but sometimes they 00:07:54.540 --> 00:07:56.190 will move towards a signal. 00:07:56.190 --> 00:07:58.690 What triggers that kind of interactions? 00:07:58.690 --> 00:08:02.580 So in looking at the cell, these are some of the older images, 00:08:02.580 --> 00:08:06.120 where certain organelles, for example, 00:08:06.120 --> 00:08:08.610 are stained so that you can see them. 00:08:08.610 --> 00:08:12.030 So peroxisomes are where degradation happens. 00:08:12.030 --> 00:08:14.340 The golgi and the ER are a part of what's 00:08:14.340 --> 00:08:16.890 known as the endomembrane system. 00:08:16.890 --> 00:08:19.980 You'll see a lot about this towards the later part 00:08:19.980 --> 00:08:22.470 of the class, where we talk about how 00:08:22.470 --> 00:08:26.310 things get outside the cell through the endomembrane 00:08:26.310 --> 00:08:27.210 system. 00:08:27.210 --> 00:08:29.850 There's the surface plasma membrane. 00:08:29.850 --> 00:08:34.470 The cytoplasm is this sort of not really aqueous-- 00:08:34.470 --> 00:08:35.850 it's an open space. 00:08:35.850 --> 00:08:37.200 But it really isn't open. 00:08:37.200 --> 00:08:39.870 It's highly congested with all kinds 00:08:39.870 --> 00:08:43.780 of molecules, all kinds of structural proteins and so on. 00:08:43.780 --> 00:08:46.950 So don't think of the cytoplasm as a solution, 00:08:46.950 --> 00:08:50.520 but think of it as a much more gel-like structure with a lot 00:08:50.520 --> 00:08:53.400 of things happening in it. 00:08:53.400 --> 00:08:57.330 The nucleus itself is also surrounded by a membrane, 00:08:57.330 --> 00:08:59.380 as is the endomembrane system. 00:08:59.380 --> 00:09:01.530 So this would be the nuclear envelope. 00:09:01.530 --> 00:09:03.480 Within the nucleus, you have a structure 00:09:03.480 --> 00:09:06.360 called the nucleolus, where aspects 00:09:06.360 --> 00:09:11.310 of the nucleic acids necessary for protein biosynthesis 00:09:11.310 --> 00:09:12.060 are made. 00:09:12.060 --> 00:09:13.620 Then there are structural proteins 00:09:13.620 --> 00:09:16.480 like microtubules and actin. 00:09:16.480 --> 00:09:18.360 But now, in this day and age, we don't 00:09:18.360 --> 00:09:20.940 have to deal with these vanilla images. 00:09:20.940 --> 00:09:23.070 We can actually use the methods that you've 00:09:23.070 --> 00:09:27.000 learned about in the last section, recombinant biology, 00:09:27.000 --> 00:09:30.070 to create new versions of proteins that 00:09:30.070 --> 00:09:34.550 have along with their sequence a marker that gives them 00:09:34.550 --> 00:09:36.600 a fluorescence-colored marker. 00:09:36.600 --> 00:09:38.700 So we are, later on in the semester, 00:09:38.700 --> 00:09:42.180 going to spend three lectures on fluorescence and cellular 00:09:42.180 --> 00:09:46.890 imaging, where you'll learn more about these fabulous proteins 00:09:46.890 --> 00:09:50.190 beyond just saying we've got a green one and a red one. 00:09:50.190 --> 00:09:52.770 We're going to give you all the background on the protein 00:09:52.770 --> 00:09:56.695 engineering that enabled those to become tools for biology. 00:09:56.695 --> 00:09:58.320 But for now, I'm just going to show you 00:09:58.320 --> 00:10:01.920 how much more interesting the images of the subcellular 00:10:01.920 --> 00:10:04.290 structures are when you've labeled, 00:10:04.290 --> 00:10:08.550 for example, a particular protein that goes exclusively 00:10:08.550 --> 00:10:12.120 to the nucleolus with a blue fluorescent protein, 00:10:12.120 --> 00:10:13.680 or to the mitochondria. 00:10:13.680 --> 00:10:15.700 Remember, Professor Martin told you 00:10:15.700 --> 00:10:17.950 we always think of these as-- and I'm not 00:10:17.950 --> 00:10:19.180 going to do the push-up. 00:10:19.180 --> 00:10:22.210 I'm just going to say it, powerhouse of the cell. 00:10:22.210 --> 00:10:26.920 I'm not doing-- [LAUGHS] I'm not great with push-ups, 00:10:26.920 --> 00:10:28.480 to be honest. 00:10:28.480 --> 00:10:32.650 But you see these sort of more tangled, extended structures. 00:10:32.650 --> 00:10:35.410 Vimentin is more of a structural protein. 00:10:35.410 --> 00:10:38.560 Here are the golgi, the endoplasmic reticulum, 00:10:38.560 --> 00:10:39.800 and the nucleus. 00:10:39.800 --> 00:10:43.150 So the colored fluorophore proteins, 00:10:43.150 --> 00:10:45.520 or the fluorescent fluorophore proteins, 00:10:45.520 --> 00:10:50.290 actually allow us, in real time, to observe dynamics. 00:10:50.290 --> 00:10:53.650 Once a protein is made, where does it go? 00:10:53.650 --> 00:10:58.300 If we add a trigger to the cell to cause an interaction, 00:10:58.300 --> 00:11:01.000 can we observe that protein, for example, 00:11:01.000 --> 00:11:03.280 migrating to the plasma membrane. 00:11:03.280 --> 00:11:06.670 Can we watch proteins being made through the ER? 00:11:06.670 --> 00:11:10.360 A variety of different things that allow us in modern biology 00:11:10.360 --> 00:11:15.110 to really look at dynamics, not just static information. 00:11:15.110 --> 00:11:18.010 And so what I'm going to talk to you about 00:11:18.010 --> 00:11:20.890 is the ways in which proteins are 00:11:20.890 --> 00:11:25.870 coded very early on in their genesis, in their biogenesis, 00:11:25.870 --> 00:11:29.860 in order to go to certain locales within the cell. 00:11:29.860 --> 00:11:34.540 So let me just give you a bit of a road map here with a protein. 00:11:38.590 --> 00:11:40.710 And where things may start-- 00:11:40.710 --> 00:11:42.410 so we have some options. 00:11:42.410 --> 00:11:46.860 Do we want to send the protein outside the cell 00:11:46.860 --> 00:11:51.660 or keep it inside the cell? 00:11:51.660 --> 00:11:54.690 Obviously, two big default differences, if you're 00:11:54.690 --> 00:11:58.770 going to go to a particular venue inside the cell. 00:11:58.770 --> 00:12:01.840 Are we going to just stay in the cytosol? 00:12:05.030 --> 00:12:07.470 That's a sort of simple-- 00:12:07.470 --> 00:12:09.510 actually, that is the default position. 00:12:09.510 --> 00:12:12.960 Because you want to remember that most proteins 00:12:12.960 --> 00:12:16.530 are made on ribosomes in the cytosol of the cell. 00:12:16.530 --> 00:12:20.100 But the statistics are that about 50% of proteins 00:12:20.100 --> 00:12:23.070 end up somewhere else than the cytoplasm. 00:12:23.070 --> 00:12:25.410 They may end up in an organelle, back 00:12:25.410 --> 00:12:27.900 in the nucleus on the surface, or secreted. 00:12:27.900 --> 00:12:28.860 So there's a lot-- 00:12:28.860 --> 00:12:32.280 so it's a good, solid 50% that don't end up 00:12:32.280 --> 00:12:36.540 staying in the cytosol, where they were originally made. 00:12:36.540 --> 00:12:39.300 Their alternative is to go to organelles. 00:12:42.720 --> 00:12:45.600 And if you're going to an organelle, remember, 00:12:45.600 --> 00:12:48.900 the ribosome is not membrane. 00:12:48.900 --> 00:12:50.850 It doesn't have a membrane perimeter. 00:12:50.850 --> 00:12:54.360 But many of the organelles do have membrane perimeters. 00:12:54.360 --> 00:12:57.090 So we're talking here about the mitochondria. 00:13:06.830 --> 00:13:08.950 That is far too long of a word. 00:13:08.950 --> 00:13:12.280 The nucleus-- so I'm going to abbreviate things 00:13:12.280 --> 00:13:18.070 like peroxisomes, or various membrane-bordered organelles, 00:13:18.070 --> 00:13:20.470 where we're going to have to figure out, if something 00:13:20.470 --> 00:13:22.840 is made in the cytoplasm, how does it 00:13:22.840 --> 00:13:25.130 get into those organelles? 00:13:25.130 --> 00:13:27.310 Now we've spoken a little bit about the fact 00:13:27.310 --> 00:13:29.790 that some proteins are made in the mitochondria. 00:13:29.790 --> 00:13:31.790 I'm going to get back to that in a moment. 00:13:31.790 --> 00:13:34.030 But all the proteins in the mitochondria 00:13:34.030 --> 00:13:36.370 are not made in the mitochondria. 00:13:36.370 --> 00:13:38.230 Some of them are shipped in. 00:13:38.230 --> 00:13:42.130 Remember the thing the endosymbiont theory, 00:13:42.130 --> 00:13:44.410 where we said that mitochondria may 00:13:44.410 --> 00:13:49.330 have originated from bacteria and been engulfed into cells. 00:13:49.330 --> 00:13:53.260 Those bacteria obviously were originally self-sufficient. 00:13:53.260 --> 00:13:55.540 But a lot of the proteins that were expressed 00:13:55.540 --> 00:13:58.720 in the mitochondria were dispensed with, 00:13:58.720 --> 00:14:01.630 and mitochondria now use proteins 00:14:01.630 --> 00:14:04.330 that are encoded by the nuclear DNA rather 00:14:04.330 --> 00:14:05.680 than the mitochondrial. 00:14:05.680 --> 00:14:09.040 But to this day, some proteins remain encoded 00:14:09.040 --> 00:14:10.730 within the mitochondria. 00:14:10.730 --> 00:14:14.920 So these are opportunities for where that may be. 00:14:14.920 --> 00:14:19.150 And I'm going to talk very specifically about signals 00:14:19.150 --> 00:14:22.540 that can get proteins into the mitochondria 00:14:22.540 --> 00:14:24.130 and into the nucleus. 00:14:24.130 --> 00:14:26.710 And it turns out that the barriers 00:14:26.710 --> 00:14:29.200 around those organelles are pretty different. 00:14:29.200 --> 00:14:30.880 I'll come back to that in a second 00:14:30.880 --> 00:14:33.310 when we get on the next slide. 00:14:33.310 --> 00:14:35.980 With respect to going outside the cell, 00:14:35.980 --> 00:14:37.550 there are two options. 00:14:37.550 --> 00:14:41.890 One option is for the protein to remain in the plasma membrane 00:14:41.890 --> 00:14:45.860 but with part of its structure outside the cell. 00:14:45.860 --> 00:14:56.900 So the other option is for the protein actually 00:14:56.900 --> 00:15:00.680 to be spit out of the cell as a soluble entity that 00:15:00.680 --> 00:15:04.340 can travel around an organism, for example, in the bloodstream 00:15:04.340 --> 00:15:05.930 and go to a remote site. 00:15:05.930 --> 00:15:08.750 And that becomes very important in signaling. 00:15:08.750 --> 00:15:13.610 So we would call those proteins secreted and soluble. 00:15:13.610 --> 00:15:16.310 So these would be membrane-bound. 00:15:16.310 --> 00:15:18.920 These would end up being soluble proteins. 00:15:18.920 --> 00:15:22.010 Let's take a look at the structure of the cell 00:15:22.010 --> 00:15:25.500 and look at where these various components are. 00:15:25.500 --> 00:15:28.880 So if you see these dots, those are free ribosomes 00:15:28.880 --> 00:15:30.350 in the cytoplasm. 00:15:30.350 --> 00:15:33.930 They would start to express different proteins. 00:15:33.930 --> 00:15:36.500 A lot of proteins are expressed in the ribosome. 00:15:36.500 --> 00:15:39.170 But in some cases, proteins become 00:15:39.170 --> 00:15:42.590 expressed on ribosomes that are associated 00:15:42.590 --> 00:15:44.930 with the endoplasmic reticulum. 00:15:44.930 --> 00:15:47.210 And therefore, you start a process 00:15:47.210 --> 00:15:50.540 whereby proteins end up being shipped 00:15:50.540 --> 00:15:52.410 to the outside of the cell. 00:15:52.410 --> 00:15:56.740 So where you see the speckles here, 00:15:56.740 --> 00:15:58.930 the free ribosome, and then the ribosomes 00:15:58.930 --> 00:16:02.200 bound to the rough endoplasmic reticulum, here, 00:16:02.200 --> 00:16:05.110 your destinies are on the right-hand side 00:16:05.110 --> 00:16:06.220 of that picture. 00:16:06.220 --> 00:16:08.920 And here, the destiny of these proteins 00:16:08.920 --> 00:16:12.190 ends up on the left-hand side of this sort of family tree 00:16:12.190 --> 00:16:13.580 that I'm showing you. 00:16:13.580 --> 00:16:16.150 There's obviously one more place where proteins are made, 00:16:16.150 --> 00:16:18.040 and that's in the mitochondria. 00:16:18.040 --> 00:16:21.130 And if you remember the first question on your exam, 00:16:21.130 --> 00:16:24.520 it described the DNA that's in the mitochondria. 00:16:24.520 --> 00:16:26.560 Going back to the endosymbiont theory, 00:16:26.560 --> 00:16:29.410 that's a circular piece of DNA. 00:16:29.410 --> 00:16:30.970 And it sets it apart. 00:16:30.970 --> 00:16:33.000 And the ribosomes in the mitochondria 00:16:33.000 --> 00:16:35.800 look more like bacterial ribosomes than you 00:16:35.800 --> 00:16:36.970 eukaryotic ribosomes. 00:16:36.970 --> 00:16:39.010 So remember, all along, we're going 00:16:39.010 --> 00:16:41.800 to try in the second half of the course 00:16:41.800 --> 00:16:44.380 to bring back knowledge we've taught you, 00:16:44.380 --> 00:16:46.390 but sort of, in a sense, endlessly 00:16:46.390 --> 00:16:49.270 remind you to keep the big picture in mind. 00:16:49.270 --> 00:16:51.670 Because we've already spoken to you about it. 00:16:57.580 --> 00:17:01.030 So this now is a nice pictorial vision 00:17:01.030 --> 00:17:02.920 of what I've just described to you. 00:17:02.920 --> 00:17:05.560 And I'm going to first of all talk about proteins 00:17:05.560 --> 00:17:07.680 that are made in the cytoplasm and may 00:17:07.680 --> 00:17:10.980 be shipped to various organelles, 00:17:10.980 --> 00:17:12.940 and how that's accomplished. 00:17:12.940 --> 00:17:14.980 And then in the second part of the class, 00:17:14.980 --> 00:17:18.250 I'll talk about how proteins are shipped to cell surface, 00:17:18.250 --> 00:17:20.290 or through expulsion from the cell. 00:17:22.810 --> 00:17:26.349 So the key mechanisms whereby proteins 00:17:26.349 --> 00:17:28.600 are trafficked to new locations are first 00:17:28.600 --> 00:17:41.710 of all using targeting sequences that are 00:17:41.710 --> 00:17:45.405 part of the protein sequence. 00:17:50.020 --> 00:17:54.340 And this is a very common way in which proteins are trafficked. 00:17:54.340 --> 00:17:55.940 They are part of the sequence. 00:17:55.940 --> 00:18:00.325 They may be at the amino or the carboxy terminus. 00:18:03.950 --> 00:18:06.810 But they are woven into the structure of your protein. 00:18:06.810 --> 00:18:10.730 So your protein comes along with a barcode saying where it's 00:18:10.730 --> 00:18:12.760 going to necessarily end up. 00:18:12.760 --> 00:18:28.430 And for the nucleus mitochondria and peroxisomes, 00:18:28.430 --> 00:18:32.690 for example, people have done extensive work 00:18:32.690 --> 00:18:37.550 with bioinformatics to basically look up protein sequences 00:18:37.550 --> 00:18:41.570 and find common themes of particular sequences 00:18:41.570 --> 00:18:44.900 that may be common to where a set of proteins may end up. 00:18:44.900 --> 00:18:46.670 Sometimes those sequences may not 00:18:46.670 --> 00:18:49.190 be easy to see just at first glance. 00:18:49.190 --> 00:18:52.640 But now there are websites that you can very, very readily 00:18:52.640 --> 00:18:55.970 put your protein sequence into the web site, and it will say, 00:18:55.970 --> 00:18:58.640 it's got a nuclear localization sequence, 00:18:58.640 --> 00:19:01.460 or a mitochondrial-targeting sequence. 00:19:01.460 --> 00:19:04.190 So we can either do this by eye or we 00:19:04.190 --> 00:19:06.380 can use informatics analysis. 00:19:06.380 --> 00:19:08.840 Informatics analysis is very valuable 00:19:08.840 --> 00:19:12.290 because sometimes information may be a bit more encrypted. 00:19:12.290 --> 00:19:14.210 And it may be a real struggle to slog 00:19:14.210 --> 00:19:16.080 through a lot of sequences. 00:19:16.080 --> 00:19:21.800 So you can really find out about the targeting sequences 00:19:21.800 --> 00:19:23.240 through bioinformatics. 00:19:25.850 --> 00:19:31.010 Because nowadays, the genomes of dozens and thousands 00:19:31.010 --> 00:19:34.010 of organisms are available readily online. 00:19:34.010 --> 00:19:36.890 And you can literally parse out information 00:19:36.890 --> 00:19:39.230 from the genomic information that gives you 00:19:39.230 --> 00:19:41.870 the proteomic information. 00:19:41.870 --> 00:19:45.560 So that's one way, so with sequences that are targeted. 00:19:45.560 --> 00:19:48.590 In some cases, those targeting sequences 00:19:48.590 --> 00:19:50.780 remain part of the protein. 00:19:50.780 --> 00:19:53.240 But in other cases, in order to ensure 00:19:53.240 --> 00:19:56.900 that the protein stays put, the targeting sequences 00:19:56.900 --> 00:19:58.050 are removed. 00:19:58.050 --> 00:20:00.230 So that's another important point. 00:20:08.580 --> 00:20:10.800 You may keep the targeting sequence, 00:20:10.800 --> 00:20:14.430 or you may lose it through the action of another enzyme that 00:20:14.430 --> 00:20:16.890 cuts off the targeting sequence when 00:20:16.890 --> 00:20:19.080 destination has been reached. 00:20:19.080 --> 00:20:24.270 Now, there's a second way that we can 00:20:24.270 --> 00:20:27.300 program where a protein may go. 00:20:27.300 --> 00:20:30.540 And these are rather useful transformations that 00:20:30.540 --> 00:20:32.560 make things even more dynamic. 00:20:32.560 --> 00:20:35.760 So let me walk you through a concept. 00:20:35.760 --> 00:20:38.580 If you think of a protein that's made on the ribosome, 00:20:38.580 --> 00:20:40.680 it's got a targeting sequence. 00:20:40.680 --> 00:20:43.410 In order to get that protein to destination, 00:20:43.410 --> 00:20:45.960 you've got to make a new batch of protein that's 00:20:45.960 --> 00:20:47.840 going to go to its destination. 00:20:47.840 --> 00:20:50.330 It's going to end up in the mitochondria. 00:20:50.330 --> 00:20:53.250 You've got to make the protein de novo. 00:20:53.250 --> 00:20:56.790 Sometimes when we need to have the action of a cell 00:20:56.790 --> 00:20:58.170 we can't wait that long. 00:20:58.170 --> 00:21:01.920 We can do things quickly and expect the cell 00:21:01.920 --> 00:21:04.950 to suddenly change what it's doing. 00:21:04.950 --> 00:21:07.320 Because we're sitting around waiting for the ribosome 00:21:07.320 --> 00:21:09.730 to make new copies of the protein. 00:21:09.730 --> 00:21:12.240 So the second way in which proteins 00:21:12.240 --> 00:21:14.970 are targeted to new destinations is 00:21:14.970 --> 00:21:17.760 through what's known as post-translational 00:21:17.760 --> 00:21:18.840 modifications. 00:21:24.370 --> 00:21:26.500 This is so unfair, Adam. 00:21:29.110 --> 00:21:30.670 I saw you using the middle boards, 00:21:30.670 --> 00:21:33.610 but it looked so much easier. 00:21:33.610 --> 00:21:37.960 So the second way to target a protein to a destination 00:21:37.960 --> 00:21:45.840 is using post-translational modification. 00:21:48.550 --> 00:21:49.730 What does this mean? 00:21:49.730 --> 00:21:52.390 What it means is that the protein is made. 00:21:52.390 --> 00:21:53.470 It's ready. 00:21:53.470 --> 00:21:54.670 It's waiting. 00:21:54.670 --> 00:21:58.480 But we haven't engaged its final destiny. 00:21:58.480 --> 00:22:01.400 We haven't triggered it to go where it needs to be. 00:22:01.400 --> 00:22:04.650 But we're waiting for an enzyme to just carry out 00:22:04.650 --> 00:22:07.540 a seemingly minor modification of that protein. 00:22:07.540 --> 00:22:10.390 And then the protein will go to its destiny. 00:22:10.390 --> 00:22:12.940 And I've shown you here examples of three 00:22:12.940 --> 00:22:14.860 types of modifications. 00:22:14.860 --> 00:22:18.760 One we will talk about today, because it's very simple 00:22:18.760 --> 00:22:21.910 to understand, lipidation. 00:22:21.910 --> 00:22:23.740 And then the other two, we'll talk 00:22:23.740 --> 00:22:32.050 about next time, phosphorylation and ubiquitination. 00:22:38.360 --> 00:22:41.790 And these are all what are known as PTMs, 00:22:41.790 --> 00:22:45.070 Post-Translational Modifications. 00:22:45.070 --> 00:22:48.960 And they are changes that occur to an amino acid side 00:22:48.960 --> 00:22:53.700 chain within an already made protein to alter its destiny. 00:22:53.700 --> 00:22:55.740 And I'd like to talk about lipidation 00:22:55.740 --> 00:23:01.080 first, because I get to remind you about cellular membranes. 00:23:01.080 --> 00:23:06.330 So remember, we've talked about these semipermeable barriers 00:23:06.330 --> 00:23:11.180 that are around organelles and around cells. 00:23:11.180 --> 00:23:13.200 And let's say that this is a membrane-- 00:23:13.200 --> 00:23:15.510 I've got to put my-- 00:23:15.510 --> 00:23:20.490 that exists between the cytoplasm 00:23:20.490 --> 00:23:23.370 and the outside of a cell. 00:23:23.370 --> 00:23:27.900 And let's say I have a protein lurking around 00:23:27.900 --> 00:23:30.920 in the cytoplasm, but I need it at the membrane. 00:23:30.920 --> 00:23:33.990 I need it to get involved in a signaling process. 00:23:33.990 --> 00:23:36.400 And I need it now to be there. 00:23:36.400 --> 00:23:38.880 If I have a soluble protein, it's not 00:23:38.880 --> 00:23:41.100 associated with the membrane. 00:23:41.100 --> 00:23:44.040 But I can use another enzyme to attach 00:23:44.040 --> 00:23:48.280 a hydrophobic, greasy tail to that protein. 00:23:48.280 --> 00:23:50.160 So what it really wants to do is to get 00:23:50.160 --> 00:23:52.080 to the hydrophobic membrane. 00:23:52.080 --> 00:23:54.750 Lipidation is such a modification. 00:23:54.750 --> 00:24:03.150 It's just the modification with a long-chain, often C16, C18, 00:24:03.150 --> 00:24:08.880 fatty acid that then renders the protein lipophilic 00:24:08.880 --> 00:24:12.750 and makes it want to move, and insert this lipophilic tail 00:24:12.750 --> 00:24:18.190 into the membrane, and part the protein of the plasma membrane. 00:24:18.190 --> 00:24:20.880 So the information is still, though, 00:24:20.880 --> 00:24:23.130 encoded within the protein. 00:24:23.130 --> 00:24:24.900 How could that happen? 00:24:24.900 --> 00:24:28.170 How could I have made that information be in the protein? 00:24:28.170 --> 00:24:30.540 What might be the strategy there? 00:24:30.540 --> 00:24:34.280 It's still encoded, but it's secret. 00:24:34.280 --> 00:24:35.310 It's cryptic. 00:24:35.310 --> 00:24:35.810 Any ideas? 00:24:41.830 --> 00:24:47.350 So I'm not going to just glom this group onto a protein. 00:24:47.350 --> 00:24:49.530 I'm going to put it somewhere specific. 00:24:49.530 --> 00:24:53.370 And so oftentimes, lipidation reactions 00:24:53.370 --> 00:24:58.140 occur site-specifically at particular sites 00:24:58.140 --> 00:25:02.400 within a sequence, and an enzyme recognizes that site 00:25:02.400 --> 00:25:05.400 and transfers the lipidic molecule to it. 00:25:05.400 --> 00:25:09.220 So lipidation actually may occur, for example, 00:25:09.220 --> 00:25:11.820 of the amino terminus of a protein. 00:25:11.820 --> 00:25:14.880 But if there are certain features within that protein, 00:25:14.880 --> 00:25:17.710 you may then attach the lipidic group. 00:25:17.710 --> 00:25:21.120 So once again, using bioinformatics, 00:25:21.120 --> 00:25:24.300 you can look at the target protein of interest 00:25:24.300 --> 00:25:28.110 and predict that it's the target of a post-translational 00:25:28.110 --> 00:25:29.790 modification reaction. 00:25:29.790 --> 00:25:32.730 So once again, the information is 00:25:32.730 --> 00:25:36.270 programmed into the sequence, but it's quite cryptic. 00:25:36.270 --> 00:25:38.970 It could be within the middle of the sequence. 00:25:38.970 --> 00:25:41.520 There could only maybe be a couple of clues. 00:25:41.520 --> 00:25:43.200 But the clues are there nonetheless 00:25:43.200 --> 00:25:47.070 that can be parsed out using computer learning and screening 00:25:47.070 --> 00:25:50.670 of sequences to say that is a target for lipidation, 00:25:50.670 --> 00:25:53.970 or phosphorylation or such. 00:25:53.970 --> 00:25:56.340 Is that clear to people? 00:25:56.340 --> 00:25:57.660 Does that make sense? 00:25:57.660 --> 00:26:01.350 The information is encoded, but you can't see that it's there. 00:26:01.350 --> 00:26:03.420 But the advantage of the post-translational 00:26:03.420 --> 00:26:06.690 modifications is that they occur on demand, 00:26:06.690 --> 00:26:10.680 as opposed to making a new protein de novo, 00:26:10.680 --> 00:26:14.160 and then having it go to a particular cellular location. 00:26:14.160 --> 00:26:17.460 Later on, when we talk about phosphorylation, 00:26:17.460 --> 00:26:19.570 you will see that phosphorylation 00:26:19.570 --> 00:26:22.800 is the bread and butter of cellular signaling. 00:26:22.800 --> 00:26:24.690 It's the light switch in every room 00:26:24.690 --> 00:26:28.110 in the cell that turns on and off in order to make functions 00:26:28.110 --> 00:26:29.700 happen within the cell. 00:26:29.700 --> 00:26:33.510 And that's a really major, dynamic post-translational 00:26:33.510 --> 00:26:37.440 modification that has significant meaning. 00:26:37.440 --> 00:26:40.650 So the reason on this little image-- 00:26:40.650 --> 00:26:42.600 I just wanted to show you the membrane 00:26:42.600 --> 00:26:46.530 and just remind you that the membrane is a supramolecular 00:26:46.530 --> 00:26:50.070 structure that's assembled with a hydrophobic core 00:26:50.070 --> 00:26:52.560 and polar head groups on both faces, 00:26:52.560 --> 00:26:55.410 as I've sort of indicated in this cartoon. 00:26:55.410 --> 00:26:57.600 So let's start with sequences that 00:26:57.600 --> 00:26:59.205 might take us to the nucleus. 00:27:02.110 --> 00:27:05.100 Now, the nuclear membrane is rather a strange entity. 00:27:10.110 --> 00:27:13.410 Because the nuclear membrane isn't a simple membrane 00:27:13.410 --> 00:27:15.220 like the plasma membrane. 00:27:15.220 --> 00:27:17.920 It's actually a double-layered membrane. 00:27:17.920 --> 00:27:20.880 So if you look at a nuclear membrane-- and I'm 00:27:20.880 --> 00:27:27.540 just going to do a job of showing a portion 00:27:27.540 --> 00:27:29.310 of the nuclear membrane. 00:27:29.310 --> 00:27:31.170 Within the nuclear membrane, there 00:27:31.170 --> 00:27:35.400 are pores, quite launch openings. 00:27:35.400 --> 00:27:39.510 And the membrane is actually a double membrane, where 00:27:39.510 --> 00:27:42.840 all of these lipid bilayers. 00:27:42.840 --> 00:27:44.265 So it's not a single membrane. 00:27:48.090 --> 00:27:56.465 It's a double membrane with large openings. 00:28:02.690 --> 00:28:05.180 And you might say, well, that's no use. 00:28:05.180 --> 00:28:08.750 There's just these great big, gaping holes in the nucleus. 00:28:08.750 --> 00:28:11.120 Anything can come and go if it wants. 00:28:11.120 --> 00:28:14.780 But the nuclear pores are kind of a special structure. 00:28:14.780 --> 00:28:18.920 Because they have a protein that's kind of disordered, 00:28:18.920 --> 00:28:21.740 that creates a tangled network. 00:28:21.740 --> 00:28:24.500 That means that that pore isn't totally open, 00:28:24.500 --> 00:28:26.150 but there's some stuff that something's 00:28:26.150 --> 00:28:29.120 got to get through to get from one side to the other. 00:28:29.120 --> 00:28:32.630 And my colleague Thomas Schwartz in biology works 00:28:32.630 --> 00:28:35.360 on the macromolecular structure of nuclear 00:28:35.360 --> 00:28:37.820 pores to understand these structures. 00:28:37.820 --> 00:28:41.060 Because these are also made through the auspices 00:28:41.060 --> 00:28:45.840 of having a lot of proteins that help create this structure. 00:28:45.840 --> 00:28:47.720 Otherwise, that membrane wouldn't 00:28:47.720 --> 00:28:53.090 stay in its proper format. 00:28:53.090 --> 00:28:56.190 So in order for a protein to get into the nucleus, 00:28:56.190 --> 00:28:58.490 if it needs to, or leave the nucleus, 00:28:58.490 --> 00:29:00.950 it has to have some kind of mechanism 00:29:00.950 --> 00:29:04.070 to get through this structure that's 00:29:04.070 --> 00:29:05.540 plugging the nuclear pore. 00:29:05.540 --> 00:29:08.580 So this would be the inside of the nucleus. 00:29:08.580 --> 00:29:11.610 And this would be the cytoplasm. 00:29:11.610 --> 00:29:15.160 So as shown on this slide, the nucleus, 00:29:15.160 --> 00:29:17.570 there's a particular protein sequence 00:29:17.570 --> 00:29:19.520 that's appended to a protein. 00:29:19.520 --> 00:29:24.120 That's known as the Nuclear Localization Sequence, or NLS. 00:29:27.510 --> 00:29:30.200 And what an NLS sequence is, it's 00:29:30.200 --> 00:29:32.750 a short sequence of amino acids that 00:29:32.750 --> 00:29:36.170 enables a protein to get to its proper destination. 00:29:36.170 --> 00:29:39.260 And these sequences are quite well recognized. 00:29:39.260 --> 00:29:42.320 They may end up being highly basic sequences. 00:29:45.860 --> 00:29:49.601 So an example of an NLS would be Lys-- 00:29:49.601 --> 00:29:51.290 it's not very exciting, but it just 00:29:51.290 --> 00:29:59.810 goes on, Lys, Lys, Lys, arginine, lysine. 00:29:59.810 --> 00:30:04.640 And it may be bounded by hydrophobic residues 00:30:04.640 --> 00:30:05.460 or other types. 00:30:05.460 --> 00:30:10.520 So that would be a typical NLS sequence that's in a protein. 00:30:10.520 --> 00:30:19.090 And I want to remind you that lysine and arginine all have 00:30:19.090 --> 00:30:21.630 side chains that at physiological pH 00:30:21.630 --> 00:30:22.970 are positively charged. 00:30:22.970 --> 00:30:25.670 So the nuclear localization sequence 00:30:25.670 --> 00:30:28.210 is something that's easily recognized because 00:30:28.210 --> 00:30:31.060 of this sort of short sequence that 00:30:31.060 --> 00:30:32.740 may be at the N- or C-terminus. 00:30:32.740 --> 00:30:34.540 I think there's either possibility. 00:30:34.540 --> 00:30:36.250 But it's a very clear sequence. 00:30:36.250 --> 00:30:38.500 You could look at your protein sequence and say, 00:30:38.500 --> 00:30:40.630 there's an NLS on that sequence. 00:30:40.630 --> 00:30:43.480 And it's the NLS sequence alone that's 00:30:43.480 --> 00:30:46.330 responsible for getting the proteins in 00:30:46.330 --> 00:30:48.250 and out of the nuclear pore. 00:30:48.250 --> 00:30:51.385 Let's mostly focus on getting into the nucleus. 00:30:55.300 --> 00:30:59.710 Basically, you have a protein structure 00:30:59.710 --> 00:31:02.560 that has an NLS sequence at one terminus. 00:31:02.560 --> 00:31:06.010 And that NLS sequence binds to another protein. 00:31:13.410 --> 00:31:15.640 Creatively, you had a little bit of chance 00:31:15.640 --> 00:31:18.250 to give proteins names in the exam. 00:31:18.250 --> 00:31:19.750 It's called importin. 00:31:19.750 --> 00:31:24.040 So it's an import protein that binds to the NLS, 00:31:24.040 --> 00:31:27.490 and as a consequence of that, will carry cargo. 00:31:27.490 --> 00:31:31.090 It will escort cargo into the nucleus of the cell. 00:31:31.090 --> 00:31:35.920 And it sends it through this meshwork of proteins. 00:31:35.920 --> 00:31:39.250 That's a very loose mesh work of proteins. 00:31:39.250 --> 00:31:41.350 And they're not ordered proteins. 00:31:41.350 --> 00:31:43.690 They're highly disordered proteins. 00:31:43.690 --> 00:31:46.455 So they make more of a filter than a plug. 00:31:46.455 --> 00:31:47.830 But they are definitely something 00:31:47.830 --> 00:31:50.500 that doesn't allow any old protein to go 00:31:50.500 --> 00:31:52.090 through that nuclear pore. 00:31:52.090 --> 00:31:55.840 NLS tags are very easy to recognize, once again, 00:31:55.840 --> 00:31:58.413 through bioinformatics analysis. 00:31:58.413 --> 00:31:59.830 And what's really cool is that you 00:31:59.830 --> 00:32:02.320 can reprogram a protein to be where you 00:32:02.320 --> 00:32:04.930 want by manipulating the NLS. 00:32:04.930 --> 00:32:07.880 So this is rather a nice set of experiments. 00:32:07.880 --> 00:32:11.290 Let's say we have a protein that we're going to micro-inject 00:32:11.290 --> 00:32:13.540 into the cytoplasm of the cell. 00:32:13.540 --> 00:32:17.080 And we want to program it to either go into the nucleus 00:32:17.080 --> 00:32:19.150 or stay outside the nucleus. 00:32:19.150 --> 00:32:21.730 That can be done readily by attaching 00:32:21.730 --> 00:32:24.940 a nuclear localization sequence to a protein 00:32:24.940 --> 00:32:29.020 along with a fluorophore dye or fluorescent protein that 00:32:29.020 --> 00:32:31.900 will allow you to observe that experiment. 00:32:31.900 --> 00:32:35.230 If you micro-inject into the cytoplasm, 00:32:35.230 --> 00:32:39.940 that protein that's got an NLS will get run into the nucleus 00:32:39.940 --> 00:32:43.250 through association of the NLS with importin. 00:32:43.250 --> 00:32:47.140 But if you chop that NLS, the protein the stuck, 00:32:47.140 --> 00:32:49.570 remains out in the cytoplasm. 00:32:49.570 --> 00:32:51.610 Let's say you want to study a new protein. 00:32:51.610 --> 00:32:54.010 I just want to show you that these NLS 00:32:54.010 --> 00:32:58.480 sequence are totally independent of the cargo they carry. 00:32:58.480 --> 00:33:01.270 You can just stick an NLS on your favorite protein 00:33:01.270 --> 00:33:03.030 who you want to interrogate. 00:33:03.030 --> 00:33:04.850 Let's take pyruvate kinase. 00:33:04.850 --> 00:33:08.140 It doesn't have anything to do with specific transport 00:33:08.140 --> 00:33:09.220 to the nucleus. 00:33:09.220 --> 00:33:11.370 But nevertheless, if you put-- 00:33:11.370 --> 00:33:14.080 if it doesn't have an NLS, it's fluorescently labeled, 00:33:14.080 --> 00:33:16.870 it stays outside in the cytoplasm. 00:33:16.870 --> 00:33:18.700 But if you put an analysis on it, 00:33:18.700 --> 00:33:21.470 you concentrate into that region of the cell. 00:33:21.470 --> 00:33:23.830 So these experiments show you that what 00:33:23.830 --> 00:33:26.020 we know about these targeting sequences 00:33:26.020 --> 00:33:30.820 can be manipulated and used to enable you to move things 00:33:30.820 --> 00:33:31.930 around in the cell. 00:33:31.930 --> 00:33:35.140 So that's one particular type of mechanism. 00:33:35.140 --> 00:33:39.400 The next mechanism I want to describe to you 00:33:39.400 --> 00:33:45.610 is the mechanism that's used for mitochondrial transports. 00:33:45.610 --> 00:33:48.055 And it's a little bit different in its strategy. 00:33:53.570 --> 00:33:58.090 So to get into the mitochondria, there is, again, a recognition 00:33:58.090 --> 00:34:07.130 sequence, in this case, a mitochondrial localization 00:34:07.130 --> 00:34:11.400 sequence that has particular characteristics. 00:34:11.400 --> 00:34:17.940 In this case, the mitochondrial localization sequence, 00:34:17.940 --> 00:34:22.310 let's say it's at the N-terminus of your protein. 00:34:22.310 --> 00:34:26.510 And it would be something that might be a mix of charges. 00:34:26.510 --> 00:34:32.750 Some Arg, Glu, Arg, Glu. 00:34:32.750 --> 00:34:35.449 So that's a typical MLS sequence. 00:34:35.449 --> 00:34:39.980 And in this case, the charge at physiological pH 00:34:39.980 --> 00:34:43.429 is different from the nuclear localization sequence, 00:34:43.429 --> 00:34:46.670 because it's an alternating positive and negative charge. 00:34:46.670 --> 00:34:48.530 So this is pretty different from this. 00:34:48.530 --> 00:34:51.179 It doesn't say bioinformatics to figure that one out. 00:34:51.179 --> 00:34:54.679 So you can then pick out mitochondrial localization 00:34:54.679 --> 00:34:55.949 sequences. 00:34:55.949 --> 00:34:59.090 And so in this case, remember, mitochondria 00:34:59.090 --> 00:35:03.140 make some of their own proteins on their circular DNA. 00:35:03.140 --> 00:35:06.710 But they've abandoned expressing all the proteins that 00:35:06.710 --> 00:35:08.540 are needed in the mitochondria. 00:35:08.540 --> 00:35:11.060 And some proteins are transported 00:35:11.060 --> 00:35:14.780 into the mitochondria using these types of sequences. 00:35:14.780 --> 00:35:16.940 But the approach, the strategy, is 00:35:16.940 --> 00:35:19.670 different from getting into the nucleus. 00:35:19.670 --> 00:35:24.710 In this case, the MLS sequence associates 00:35:24.710 --> 00:35:28.520 with a protein channel that is in a closed state. 00:35:28.520 --> 00:35:30.090 So here's a membrane. 00:35:30.090 --> 00:35:31.790 Here's the makings of a channel. 00:35:31.790 --> 00:35:33.530 But it's in a closed state. 00:35:33.530 --> 00:35:37.370 But once the protein with the NLS sequence binds to that, 00:35:37.370 --> 00:35:39.830 that channel opens. 00:35:39.830 --> 00:35:42.320 It's triggered by the binding of that sequence 00:35:42.320 --> 00:35:47.420 to a portion of the protein that's outside that membrane. 00:35:47.420 --> 00:35:51.590 And that then allows the protein to be unfolded and transported 00:35:51.590 --> 00:35:55.940 into the mitochondria, where that sequence may be removed. 00:35:55.940 --> 00:35:59.070 And then protein refolds in the mitochondria. 00:35:59.070 --> 00:36:01.070 So it's a very different strategy 00:36:01.070 --> 00:36:03.860 for that and the nuclear localization sequence. 00:36:03.860 --> 00:36:07.850 So you'll find, for many different organelles 00:36:07.850 --> 00:36:11.900 in the cell, there might be very specific localization sequences 00:36:11.900 --> 00:36:14.250 that you could look up and learn about. 00:36:14.250 --> 00:36:16.220 But one thing I want to mention to you 00:36:16.220 --> 00:36:19.850 is that these localization details are very important. 00:36:19.850 --> 00:36:23.840 And many diseases in cells are a consequence 00:36:23.840 --> 00:36:27.570 of proteins not being localized to the right place. 00:36:27.570 --> 00:36:30.260 If you're not in the right place at the right time, 00:36:30.260 --> 00:36:32.510 then things will start to go wrong 00:36:32.510 --> 00:36:35.730 with the signaling or the processes of the cell. 00:36:35.730 --> 00:36:39.470 So diseases are frequently associated 00:36:39.470 --> 00:36:41.360 with mislocalization. 00:36:41.360 --> 00:36:48.450 So now what we're going to do is basically say, 00:36:48.450 --> 00:36:51.720 we've taken care of understanding things made 00:36:51.720 --> 00:36:52.680 in the cell. 00:36:52.680 --> 00:36:54.570 They either stay in the cytosol or they'll 00:36:54.570 --> 00:36:58.950 go to organelles based on particular types of strategies 00:36:58.950 --> 00:37:03.450 that are largely dependent on short tagging sequences, 00:37:03.450 --> 00:37:07.380 but in other cases, may be dependent on post translational 00:37:07.380 --> 00:37:09.360 modification. 00:37:09.360 --> 00:37:09.860 All right. 00:37:09.860 --> 00:37:12.990 So here is a cartoon. 00:37:12.990 --> 00:37:16.020 But actually, I want to do something slightly different 00:37:16.020 --> 00:37:19.530 if it doesn't take too long. 00:37:19.530 --> 00:37:22.710 Now, when we first talked about translation 00:37:22.710 --> 00:37:26.010 on the ribosome, what you see there in green and yellow 00:37:26.010 --> 00:37:27.210 is the ribosome. 00:37:27.210 --> 00:37:29.790 The dark band is a messenger RNA. 00:37:29.790 --> 00:37:32.550 The dark blue are transfer RNAs that 00:37:32.550 --> 00:37:37.050 are being helped with elongation factors to get to the ribosome. 00:37:37.050 --> 00:37:38.910 But what I want to point out here 00:37:38.910 --> 00:37:42.420 is the emerging sequence of polypeptide 00:37:42.420 --> 00:37:44.970 coming out through a tunnel on the ribosome. 00:37:44.970 --> 00:37:49.710 Now, if a protein is going to be destined outside the cell, 00:37:49.710 --> 00:37:53.050 it is expressed with what's known as a signal sequence. 00:37:53.050 --> 00:37:55.380 It's about a 20-amino acid residue 00:37:55.380 --> 00:37:59.400 sequence that is recognized by the signal recognition 00:37:59.400 --> 00:38:00.630 particle. 00:38:00.630 --> 00:38:04.290 And then translation slows down and clamps 00:38:04.290 --> 00:38:06.990 the ribosome on the endoplasmic reticulum 00:38:06.990 --> 00:38:10.350 membrane so that the new peptide starts 00:38:10.350 --> 00:38:14.010 being threaded into the endoplasmic reticulum 00:38:14.010 --> 00:38:16.680 through what's known as the translocon. 00:38:16.680 --> 00:38:21.120 So you're now not sending the protein out to the cytoplasm, 00:38:21.120 --> 00:38:23.400 but you're rather sending the protein 00:38:23.400 --> 00:38:26.050 into the endoplasmic reticulum. 00:38:26.050 --> 00:38:31.050 And you're also sending it down this branch of the protein 00:38:31.050 --> 00:38:32.760 biosynthesis pathway. 00:38:32.760 --> 00:38:35.400 You see this piece of protein emerging. 00:38:35.400 --> 00:38:39.510 This hatched portion is the cytoplasm. 00:38:39.510 --> 00:38:42.720 The gray portion is the endoplasmic reticulum. 00:38:42.720 --> 00:38:46.470 So there is a complex machinery at play 00:38:46.470 --> 00:38:50.460 that enables proteins to be made in the cytoplasm 00:38:50.460 --> 00:38:53.772 but now targeted to a completely new location. 00:38:53.772 --> 00:38:55.230 And these are the proteins that are 00:38:55.230 --> 00:38:58.980 going to be destined to either stay in the plasma membrane 00:38:58.980 --> 00:39:01.050 or be secreted from the cell. 00:39:01.050 --> 00:39:04.680 And this view here gives you a little bit more 00:39:04.680 --> 00:39:05.910 than the cartoon. 00:39:05.910 --> 00:39:08.640 So ribosome-- a signal peptide is 00:39:08.640 --> 00:39:11.490 made that is a green peptide sequence that's 00:39:11.490 --> 00:39:13.890 about 20 amino acids long. 00:39:13.890 --> 00:39:17.160 That is actually called a signal peptide. 00:39:17.160 --> 00:39:19.500 It's signaling for synthesis through 00:39:19.500 --> 00:39:21.750 the endomembrane network. 00:39:21.750 --> 00:39:26.160 That causes the ribosomes to dock down on the cytosol ER 00:39:26.160 --> 00:39:29.040 membrane and keep on being synthesized 00:39:29.040 --> 00:39:32.880 so that proteins are made into that endomembrane system. 00:39:32.880 --> 00:39:35.190 And you can think of this cavernous endomembrane 00:39:35.190 --> 00:39:39.510 system as your tunnels out of a cell for either display 00:39:39.510 --> 00:39:43.170 on the surface of the cell or for secretion entirely 00:39:43.170 --> 00:39:44.350 in vesicles. 00:39:44.350 --> 00:39:47.550 So let's take a look at how that occurs. 00:39:47.550 --> 00:39:49.920 When you make a protein in that way, 00:39:49.920 --> 00:39:53.400 see the dark dots, the rough ER? 00:39:53.400 --> 00:39:57.180 These are ribosomes that are attached to the membrane. 00:39:57.180 --> 00:39:59.550 Proteins are made into the membrane. 00:39:59.550 --> 00:40:03.120 And then the endomembrane system is not really 00:40:03.120 --> 00:40:06.540 just a tunnel or a labyrinth. 00:40:06.540 --> 00:40:08.610 But actually, each of those layers 00:40:08.610 --> 00:40:12.120 spits off vesicles that fuse with next layers 00:40:12.120 --> 00:40:15.840 to gradually make their way outside of the cells. 00:40:15.840 --> 00:40:17.850 So here you see there are vesicles. 00:40:17.850 --> 00:40:20.430 You're always keeping proteins associated 00:40:20.430 --> 00:40:24.310 with membrane as you go through the endomembrane system. 00:40:24.310 --> 00:40:27.120 And here is a vesicle that's got protein in it. 00:40:27.120 --> 00:40:30.570 It may either release it to the outside of the cell, 00:40:30.570 --> 00:40:34.650 or the protein may be associated with the membrane 00:40:34.650 --> 00:40:38.760 of the vesicle and stay parked in the plasma membrane. 00:40:38.760 --> 00:40:42.990 And so I just want to give you one final slide where 00:40:42.990 --> 00:40:46.310 I talk about the biogenesis of membrane proteins. 00:40:46.310 --> 00:40:48.180 Now, this is pretty complicated stuff. 00:40:48.180 --> 00:40:51.540 Because you have to remember what's inside and out. 00:40:51.540 --> 00:40:56.490 So I spent more time than I should have on this cartoon 00:40:56.490 --> 00:40:59.430 to show you which end of the protein 00:40:59.430 --> 00:41:02.100 ends up outside the cell and which inside the cell, 00:41:02.100 --> 00:41:05.490 and how you make multi-membrane-spanning 00:41:05.490 --> 00:41:06.130 proteins. 00:41:06.130 --> 00:41:09.030 So let's take a look at this in detail now, looking-- 00:41:09.030 --> 00:41:10.620 here's the ribosome. 00:41:10.620 --> 00:41:12.720 Here's the protein emerging. 00:41:12.720 --> 00:41:14.580 If there's signal sequence there, 00:41:14.580 --> 00:41:17.280 that ribosome docks down on the membrane 00:41:17.280 --> 00:41:21.300 and starts translating the protein, amino terminus 00:41:21.300 --> 00:41:23.730 first, into the endoplasmic reticulum. 00:41:23.730 --> 00:41:25.800 We'll all OK with that. 00:41:25.800 --> 00:41:30.576 As synthesis continues, we may reach the stop codon 00:41:30.576 --> 00:41:32.640 on the messenger RNA. 00:41:32.640 --> 00:41:35.820 And what may happen is that the protein may remain 00:41:35.820 --> 00:41:37.710 associated with membrane. 00:41:37.710 --> 00:41:40.020 The amino terminus will be in the ER. 00:41:40.020 --> 00:41:42.910 And the C-terminus will remain on the other side. 00:41:42.910 --> 00:41:46.650 There are a number of different configurations. 00:41:46.650 --> 00:41:50.490 But if we want to start to transport this protein 00:41:50.490 --> 00:41:53.280 to the surface of the cell, that will then 00:41:53.280 --> 00:41:55.890 stay associated with membrane but not 00:41:55.890 --> 00:41:59.330 in the form of the flat membrane that it was delivered into. 00:41:59.330 --> 00:42:04.320 But that membrane may pinch off into a spherical vesicle. 00:42:04.320 --> 00:42:06.720 But you still have the C-terminus outside 00:42:06.720 --> 00:42:09.000 and the N-terminus inside. 00:42:09.000 --> 00:42:11.730 That will then work its way through 00:42:11.730 --> 00:42:16.110 the endomembrane system, and ultimately, fuse 00:42:16.110 --> 00:42:17.190 with the cytosol. 00:42:17.190 --> 00:42:20.190 This is the really fun part. 00:42:20.190 --> 00:42:22.305 And then, once it's fused with the cytosol, 00:42:22.305 --> 00:42:24.660 it has the option to be displayed 00:42:24.660 --> 00:42:26.410 on the outside of the cell. 00:42:26.410 --> 00:42:26.910 Why? 00:42:26.910 --> 00:42:28.620 You have a protein. 00:42:28.620 --> 00:42:30.450 The N-terminus is on the outside. 00:42:30.450 --> 00:42:32.610 The C-terminus is on the inside. 00:42:32.610 --> 00:42:36.090 So that shows you the biogenesis of the cell surface 00:42:36.090 --> 00:42:38.130 protein that's stuck in the membrane 00:42:38.130 --> 00:42:41.580 through its membrane-associated domain. 00:42:41.580 --> 00:42:43.900 If you're not going to stay with the membrane, 00:42:43.900 --> 00:42:46.530 you can actually also simply release this 00:42:46.530 --> 00:42:50.010 into the vesicle for release of a soluble protein. 00:42:50.010 --> 00:42:51.330 I will not go through this. 00:42:51.330 --> 00:42:53.940 But there are miraculous steps that 00:42:53.940 --> 00:42:58.320 end up in the biogenesis of multi-transmembrane proteins. 00:42:58.320 --> 00:43:00.870 Because each of those transmembrane domains 00:43:00.870 --> 00:43:04.400 gets made in the translocon and gets shuttled sideways. 00:43:04.400 --> 00:43:07.620 And you start piling up transmembrane domains 00:43:07.620 --> 00:43:09.060 that span the membrane. 00:43:09.060 --> 00:43:10.800 And in the next class, we're going 00:43:10.800 --> 00:43:15.090 to see how useful these proteins are in cellular signaling. 00:43:15.090 --> 00:43:18.990 So those are very important proteins to think about. 00:43:18.990 --> 00:43:22.020 One last thing-- so let's think about this. 00:43:22.020 --> 00:43:27.090 For either configuration, either post-translational modification 00:43:27.090 --> 00:43:31.440 or using targeting sequences, when do we define where 00:43:31.440 --> 00:43:33.660 the protein's going to end up? 00:43:33.660 --> 00:43:35.625 Where's the information first defined? 00:43:39.020 --> 00:43:42.730 Anyone want to answer me and explain why? 00:43:42.730 --> 00:43:43.424 Yes? 00:43:43.424 --> 00:43:45.400 AUDIENCE: Would it be B, the mRNA sequence, 00:43:45.400 --> 00:43:47.353 because that would have a significant portion 00:43:47.353 --> 00:43:48.020 of the splicing? 00:43:51.910 --> 00:43:53.490 BARBARA IMPERIALI: It's a good try. 00:43:53.490 --> 00:43:56.760 But you want to remember, yes, splicing is important. 00:43:56.760 --> 00:43:59.400 But when was the sequence actually 00:43:59.400 --> 00:44:00.660 in the entire pre-mRNA? 00:44:03.773 --> 00:44:05.190 When would that have been defined? 00:44:07.980 --> 00:44:09.040 Yeah? 00:44:09.040 --> 00:44:09.540 Sorry. 00:44:09.540 --> 00:44:10.350 Carmen? 00:44:10.350 --> 00:44:13.570 AUDIENCE: Is it in the genomic DNA sequence? 00:44:13.570 --> 00:44:14.560 BARBARA IMPERIALI: Yes. 00:44:14.560 --> 00:44:18.040 Because you never have information in the RNA 00:44:18.040 --> 00:44:19.490 that wasn't in the DNA. 00:44:19.490 --> 00:44:21.790 So the DNA has got the information there. 00:44:21.790 --> 00:44:24.880 Yeah, it may need a bit of splicing to put things 00:44:24.880 --> 00:44:26.390 in the right place. 00:44:26.390 --> 00:44:28.780 But the information is there in the DNA. 00:44:28.780 --> 00:44:33.280 So you want to remember, for all of this targeting information, 00:44:33.280 --> 00:44:36.310 it's in the genomic information most commonly. 00:44:36.310 --> 00:44:37.870 It's the genomic information that 00:44:37.870 --> 00:44:41.050 has the patterns of sequences for post-translational 00:44:41.050 --> 00:44:42.150 modification. 00:44:42.150 --> 00:44:44.470 It's the genomic information that has 00:44:44.470 --> 00:44:46.990 things like NLSes and MLSes. 00:44:46.990 --> 00:44:48.320 They're already there. 00:44:48.320 --> 00:44:50.350 But they are often encrypted. 00:44:50.350 --> 00:44:52.510 And there was a very nice point there, though. 00:44:52.510 --> 00:44:58.240 If you want to send to make a single chunk of a genome that 00:44:58.240 --> 00:45:02.350 encodes either a protein that's going to be exported 00:45:02.350 --> 00:45:06.430 through the secretory pathway or stay in the cytosol, 00:45:06.430 --> 00:45:10.120 you might splice in or out a signal sequence. 00:45:10.120 --> 00:45:13.750 So that's a really good way, using the same original DNA 00:45:13.750 --> 00:45:16.420 sequence, to actually get to proteins 00:45:16.420 --> 00:45:19.870 that fulfill different final destinies within the cell. 00:45:19.870 --> 00:45:22.120 So next time, we're going to talk about signaling. 00:45:22.120 --> 00:45:24.240 It's going to be a blast.