MIT OpenCourseWare http://ocw.mit.edu 7.012 Introduction to Biology, Fall 2004 Please use the following citation format: Eric Lander, Robert Weinberg, and Claudette Gardel, 7.012 Introduction to Biology, Fall 2004. (Massachusetts Institute of Technology: MIT OpenCourseWare). http://ocw.mit.edu (accessed MM DD, YYYY). License: Creative Commons Attribution-Noncommercial-Share Alike. Note: Please use the actual date you accessed this material in your citation. For more information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/termsMIT OpenCourseWare http://ocw.mit.edu 7.012 Introduction to Biology, Fall 2004 Transcript – Lecture 14 Good morning. Good morning. Today I'm going to talk about, this is lecture 14, and I'm going to talk about protein localization. Now, some of you may remember that earlier in the semester I was walking around with this sling. And so to help me from writing on the board, even though it is this arm, I have made a PowerPoint presentation of most of the things that I would have written on the board. And for your ease and comfort, this PowerPoint presentation will be posted online so you won't have to write down everything in my slides. So just sort of sit back. And I will write a few things on the board, so you can write those down. OK. So now you guys have heard about central dogma from Professor Eric Lander and you've heard about gene regulation last Friday. Here is something that you're familiar with, this image here depicting central dogma. DNA replicates to DNA. This is replication. Replication. DNA is translated, excuse me, transcribed to RNA. Transcription. And RNA is translated to protein. Central dogma. Where does this occur? Where does replication occur in a cell? Nucleus. Good. Where does transcription occur in a cell? Nucleus. I heard nucleus. That is correct. And where does translation occur? Cytoplasm. Ah, and yet we know that these processes require proteins to do them. OK. You've just described where these processes occur in a eukaryotic cell. Let's say you're a bacterium. In bacteria, where does replication, transcription and translation occur? Where? Cytoplasm. OK. So now we've made bacteria look very simple. But they're not that simple. And so let's take a look here. Here's a bacterial cell. I've drawn what could be E. coli. It has an outer membrane, an inner membrane, and the space in between is the perisplasm. Now, here is its circular chromosome. I've transcribed some gene to an RNA and a ribosome will pop on and make a protein which is in the cytoplasm.Yet some proteins are localized to the inner membrane, others are localized to the periplasm, and some are localized to the outer membrane, and others are actually exported completely outside the cell. Even more complicated is a eukaryotic protein, because not only does it have a plasma membrane where proteins are localized. It has a bunch of organelles. There's the nucleus and there's mitochondria and there's endoplasmic reticulum and Golgi apparatus. And it, too, translates RNA by ribosomes in the cytoplasm. So how do these proteins get back to the nucleus or go into the mitochondria or get into the organelles? So what we're going to do in the next few slides is we're going to follow the process, because they're so similar in bacteria and eukaryotic cells, of how proteins get to the membrane and how they get outside the cell. And then I'll go back and talk about how proteins get into some of the organelles. So let me show you what some of the proteins are. So an example of a cytoplasmic protein in bacteria is beta galactosidase. You've heard about it. It breaks down lactose. It's in the cytoplasm. An example of a membrane protein is a lactose receptor. The lacY permease that's on the surface of the cell brings lactose in. An example of a fully secreted protein is a toxin. For instance, bacillus anthracis makes anthrax toxin. It's completely exported from the cell. In a eukaryotic cell there's a bunch of cytoplasmic proteins. There are all of the glycolytic enzymes. And, for instance, biosynthetic amino acid enzymes like histidine synthesis enzymes, those are cytoplasmic. For a membrane protein there are receptors, like the receptor for insulin, a hormone, a peptide hormone, or growth factor receptors, every receptor that's membrane-bound. And a fully secreted protein. Some cells like pancreatic cells secrete insulin. Some cells like some of your immune cells secrete antibodies. OK. So it was not clear how these cytoplasmically made proteins, proteins that were made in the cytoplasm got to this location. And the person who worked on this was George Palade. And this was in the fifties. And he studied pancreatic cells because they're master secretors. And he was able to perfect his microscopic technique. And you can see here this is a pancreatic cell. This is endoplasmic reticulum studded with ribosomes. These are mitochondria. This is the nucleus. Here is another picture that hetook. And here is the rough endoplasmic reticulum studded with ribosomes. Here's Golgi apparatus. And then there's, like, little vesicles. So he did this experiment where he decided he would pulse-label proteins as they were being synthesized in a pancreas, directly in a pancreas. So what he did was he injected radioactive amino acids directly into the pancreas of hamsters. I guess I could draw a little hamster here. And he directly injected radioactive isotopes. And what he's doing is these radioactive amino acids will be incorporated into proteins as they're being translated, and he can follow the population of freshly translated proteins through the cell. So he injects hamsters with the radioactive amino acids. And then at various time points he adds, he also injects glutaraldehyde. So first the label, then glutaraldehyde. And what this does is it fixes the cells in its tracks. Whatever the cell is doing it just stops. And he removes the pancreas and he looks at the cells. This fixes the cells. So, Tom, I don't know what's going on here. Can we not use this, Tom? All right. It's just doing it on its own. It has some time thing? Oh. All right. So what he found was at the early time points, now, what I did, I did this, OK? He didn't see yellow. What I did was I added yellow to his original slide to show you at the earliest time point he found the label associated with the endoplasmic reticulum. At the
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