C2006/F2402 '07 -- Outline For Lecture #6
(c) 2007 Dr. Deborah Mowshowitz , Columbia University, New York, NY. Last update 01/31/2007 10:48 AM
Handouts: 5C.RME 6A: Protein Transport & Structure of Capillaries & Transcytosis (Not on web. Click here for a diagram of a capillary, and here for a diagram of transcytosis. ) 6B -- Signal Hypothesis -- Co-translational Import
I. RME -- Receptor Mediated Endocytosis, cont.
A. Review of Early Stages of Cycle -- (Numbers match steps on handout 5C.) Click here for animation. How uncoated vesicle forms.
(1). Receptors bind material(ligand) to be internalized
(2). Receptors are in (or migrate to) coated pits (clathrin-coated parts of membrane)
(3). Membrane starts to invaginate to form coated vesicle. A single vesicle can contain more than one type of receptor plus ligand.
(4). Coated vesicle forms(pinching off of vesicle may be an energy requiring step)
(5). Uncoating occurs relatively quickly(uncoating requires energy)
B. How Sorting Occurs in Endosome
(6). Vesicle is acidified to become endosome (or fuses with pre-existing endosome), and sorting of receptor(s) and ligand(s) begins.
- A single endosome may contain many different receptors and ligands, and different ones are sorted differently. (Some examples are given in detail below.)
- The uncoated, acidified vesicle can be called an endosome, early endosome, or a sorting vesicle.
- Acidification requires energy to run proton pump -- to move H+ into vesicle at expense of ATP. Pump is in membrane of vesicle.
Note: Details of sorting and recycling -- the remaining steps -- vary with material endocytosed. More details below for individual cases.
(7). Endosome splits. The substance we are following, and/or its receptor, can end up in either half.
In example shown on handout, one half gets the receptor and one half gets the ligand, as is the case for LDL Other examples will be discussed in class and are outlined in detail below.
Note: endosome may not simply split in one step; process of sorting may be gradual. Pieces of different composition may gradually bud off as internal composition of remainder changes.
C. What Happens to the Different Parts of the Endosome?
(8A). Fate of vesicle with materials to be recycled (receptors and/or carriers) -- this vesicle fuses with plasma membrane. (In case of LDL, this vesicle would contain the receptor for LDL.)
(8B). Fate of vesicle with material that was endocytosed -- Vesicle delivers contents to appropriate intracellular compartment. (For LDL, delivers to lysosomes, so material is degraded.)
(9). Exocytosis occurs-- returns receptors and/or other components to the plasma membrane or outside of cell.
Try Problem 2-6.
D. Some Specific Examples1. LDL (Low density lipoprotein) -- more details. See Becker, Box 12B or Purves 5.16 (5th ed.). 6th & 7th ed. of Purves don't have pictures, but do have info in text. Many of LDL details will be included in general case. Click here for a picture of LDL.
a. What is it? A particle containing cholesterol esters + some other lipids + a protein (carrier). Particle contains esterified cholesterol covered by monolayer of amphipathic lipid (phospholipid plus some unesterified cholesterol) + one molecule of carrier protein.
2. EGF (Epidermal Growth Factor)
b. Why LDL?
(1) Why a monolayer on outside? Solubility. Cholesterol is insoluble in blood. (Too hydrophobic.) Need a way to ferry cholesterol through blood and into cell -- Cholesterol transport requires formation of particle with hydrophilic surface
(2) Why a carrier protein ? For binding to cell surface receptor (LDL receptor). A protein needed as ligand to bind to receptor.
(3) Summary of Roles of Parts of LDL:
(1). Carrier Protein = Ligand = what actually binds LDL receptor = protein part of LDL .
(2). Cholesterol -- What cell actually needs is the cholesterol part (for building its membranes &/or hormone synthesis).
c. Receptor, but not carrier, is recycled. Note: there are 2 separate proteins here that are easily confused
(1) Receptor protein on the cell surface = LDL receptor = binds LDL and allows uptake of cholesterol
(2) Carrier protein = ligand for LDL receptor = part of LDL and helps carry cholesterol through the blood.
d. Receptor and carrier are separated inside sorting vesicles/endosomes. All of LDL goes with carrier protein.
e. Need lysosomes to degrade carrier and release cholesterol (cholesterol esters in LDL must be split for cholesterol to be used).
(1). How LDL reaches lysosomes: vesicles/endosomes holding substrate fuse with pre-existing lysosomes, or vesicles with substrate fuse with vesicles from Golgi carrying newly made hydrolases to form new lysosomes. (More details on how hydrolases pass through the Golgi and are targeted to lysosomes to be discussed later.)
(2) Current terminology: relationship of early endosomes, late endosomes & lysosomes. Note: Most of this is FYI. In this course, the term "endosomes" will be used for both early and late endosomes.
(a). Early endosome = sorting vesicle. Term is used differently by different authors. Can be "early" on pathway into cell (by endocytosis) and/or "early" on pathway from Golgi to lysosomes. Therefore, early endosomes can mean any of the following:
(i) Uncoated & acidified vesicles from invagination of plasma membrane carrying newly endocytosed material,
(ii). Vesicles coming from Golgi carrying newly made hydrolases (more on this later).
(iii) Vesicles formed by fusion of (i) + (ii).
(b). Late endosome = vesicle containing hydrolytic enzymes (not yet activated) plus potential substrate. More acidic than early endosome. Material not destined for lysosomes has been jettisoned.
(c). Lysosomes = vesicle containing active hydrolytic enzymes and substrate. More acidic than late endosome. Formed by maturation of late endosome and/or fusion with pre-existing lysosome.
(3). Older terminology found in some texts (FYI only):
(a). Primary lysosome = vesicle with enzymes only.
(b). Secondary lysosome = enzymes + substrate = result of fusion of primary lyso. + another vesicle containing substrate.
f. Function of LDL uptake -- to supply a nutrient (cholesterol).
a. No carrier required; EGF (a protein -- unlike cholesterol, or Fe, see below) binds to receptor; EGF = signaling molecule = ligand for cell surface receptor & substance that will be transported into the cell.
b. Function of uptake -- to regulate signaling -- turn off signal and down regulate receptors (reduce # of cell surface receptors).
c. Receptor not recycled -- Ligand (signal molecule) and receptor degraded together.
d. Need lysosomes (to degrade both receptor and ligand).
a. What is transferrin? Fe needs carrier protein (like cholesterol does) for transport and binding to receptor; carrier (= ligand for cell receptor) is called transferrin.
b. Both carrier & receptor are recycled.
c. No lysosomes needed -- iron diffuses out of endosome (using carrier protein or channel in membrane?); no protein is degraded.
d. Carrier and receptor separate outside cell after recycled
(1). Fe/transferrin binds to receptor at neutral pH and enters cell by RME.
(2). Inside cell, Fe diffuses out of vesicle into cytoplasm, leaving apo-transferrin stuck to receptor ("apo" means without ligand or cofactor).
(3). Apo-transferrin (= transferrin without Fe) sticks to receptor at low pH (in endosome) but separates at neutral pH (outside cell). This is contrary to usual behavior -- Most ligands stick to receptors at neutral pH but separate at low pH found in endosome.
(4). Note that apo-transferrin and Fe/transferrin have different affinities for the receptor at neutral pH. Under these conditions (neutral pH), Fe/transferrin binds to the receptor, and apo-transferrin separates from the receptor.
e. Function of uptake -- to supply a nutrient (Fe).
D. For Reference: Compare & Contrast for the examples described above for transport of X
|What's carried in (what is X)?||Fe||Cholesterol||Growth Factor|
|Function of X||Metabolism (Fe is cofactor for many proteins)||Metabolism (cholesterol is a component of cell membranes; used for hormone synthesis)||Signal|
|Ligand (What binds receptor?)||Transferrin||LDL (Carrier protein part)||EGF|
|Ligand/Carrier protein separate from X?||Yes||Yes||No|
|Do ligand & receptor separate inside cell?||No||Yes||No|
|Where do ligand & receptor separate?||Outside the cell||In endosomes||Not separated -- both degraded|
II. Structure of Capillaries & Transcytosis -- see handout 6A. Pictures are provided on handout since function is hard to understand without the anatomy.
A. Capillary Structure. Shows how endothelial cells surround capillary lumen, and pores between cells allow diffusion (of glucose and other small molecules, ) in and out of capillary. Proteins are too big to fit through pores.
B. Transcytosis -- variant of RME. Material binds to receptor and is endocytosed on one surface of the cell. Vesicle moves across cell and material is exocytosed on a different surface. Can be used to move proteins, such as some antibodies, across epithelia, (as shown on diagram of transcytosis*) or to move proteins from inside capillary to outside (as shown on handout). Note transcytosis requires a receptor for each substance transported.
(*picture shows caveoli, which are a specialized type of endocytotic vesicle)
III. Labeling -- How do you follow material coming in (or going out) of the cell?
A. Types of Labeling (using added tracers)
B. Detection -- How do you find where the radioactivity is?
1. Continuous Labeling -- switch from regular, ordinary material to labeled material (material containing radioactivity, fluorescence, etc.) and follow what gets labeled first (with radioactivity, fluorescence, etc.), what gets labeled next, and so on. We will discuss radioactive labeling, but the principle is the same whether label is radioactivity, fluorescence, etc.
2. Pulse-Chase Experiments -- supply radioactive material for a brief time (pulse) and then switch back to ordinary, non-radioactive material (chase). Follow where the radioactivity goes. The "pulse" passes through the cell like a mouse through a boa constrictor. Just as different parts of the boa constrictor bulge out temporarily as the mouse passes down the snake, so different parts of the cell become radioactive temporarily, one at a time, as the radioactive material passes through. Then as the "pulse" or the "mouse" passes on, each part will return to normal -- non radioactive or normal size, depending on whether we are referring to the cell or to the snake.
1. Autoradiography -- Cover a layer of labeled cells with photographic emulsion and count radioactive grains over each organelle or part of the cell. See Becker, Appendix, A-18 or Guide to Microscopy. This is similar to doing in situ assays, in that you examine intact cells to pin down the location of what you are looking for.
Note: Becker's Appendix (or Guide to Microscopy in 5th ed.) has a lot of useful background info on microscopic methods, including immunofluorescence, freeze fracture, etc.
2. Fractionate First -- Break up labeled samples, fractionate into various organelles, and measure radioactivity in each fraction. This is similar to the "grind and find" procedure, in that you break up the cells, separate them into their parts, and test a solution or suspension of each part for what you are looking for.
C. An example: How do you follow newly made molecules moving through the cell and/or on their way out? How do we know newly made proteins go from RER to Golgi etc.?
In examples of detection above, emphasis was on following molecules going in to the cell. This example is about following newly made molecules on their way out.
1. General idea -- Add labeled precursors (small molecules) and measure incorporation into macromolecules.
a. Add labeled precursors, and take cell samples after increasing time intervals.
b. For each sample, wash out unused ('unincorporated') small molecules -- removes labeled molecules not used for synthesis so not incorporated into macromolecules. Radioactivity remaining in dif. parts of the cell is in macromolecules.
c. Use autoradiography for measurement of radioactivity in each cell part or isolated fraction.
2. A specific example-- following secreted proteins out.
a. Continuous label vs pulse-chase results (I will draw sample curves on board; see also fig. 12-10 of Becker. )
b. Implications: newly made proteins to be secreted go → RER → Golgi → secretory vesicles → outside (See Becker fig. 12-8.) Click here for animation.
To review labeling and RME, try problems 2-8 & 2-11; by now you should be able to do all the problems in problem set 2. For another example of the use of labeling, try 3-2D.
D. Another Type of Labeling -- Cell makes its own labeled protein using GFP
GFP = green fluorescent protein = small fluorescent protein made by jelly fish. (Click here for picture of GFP.) Used as tag to follow proteins inside the cell. GFP is not added from outside. Instead, genetic engineering used to splice gene for GFP to gene for protein of interest. Recombinant gene makes a fusion protein = normal sequence of amino acid + GFP. Fusion protein (including GFP) is made internally by the cell; in other words, cell makes its own fluorescently tagged version of the protein. Protein usually works normally, but location of protein can be easily followed in cell, because protein has GFP attached. GFP labeled protein is used for many purposes, including following newly made protein through the cell.
GFP is described in 6th ed. of Purves p. 324; picture of its use is on p. 885. See also Becker A-10 (Guide to Microscopy.)
GFP is often used to identify cells that express (turn on) a particular gene. For an example see this picture. The cells that "light up" are the only ones that express (turn on) the fusion gene. Only these cells produce a fusion protein containing GFP. (This example also illustrates why people use small, transparent organisms as "model organisms.") For a really startling picture, try this one. See the bottom of the page for an explanation.
See problem 2R-4 for an example
of the use of GFP labeling.
IV. Sorting of Proteins to their Proper Place: Overview (See handout 6A & Becker fig. 22-14 (20-14) or Purves 12.14 -- terminology in Purves is slightly different.)
A. What determines the fate (final location) of each individual protein? The amino acid sequence of the protein. The ability of each protein to reach its proper destination is built into the protein itself. The presence (or absence) of localization signals in the amino acid sequence is the determining factor.
1. What's a localization signal: a group of amino acids acting as an "address" or "tag" directing the protein to a particular destination.
2. Terminology: The localization signal or "tag" is often called a localization sequence (LS) or patch.
a. LS -- if it consists of a continuous section in the peptide chain.
b. Patch -- if it consists of a contiguous section in the folded protein. (But AA are not next to each other in the unfolded chain.)
3. Use of tags: The localization sequence/patch directs the protein to the ER, nucleus, etc. Several different localization sequences, which are read sequentially, may be needed to direct a protein to its proper destination. If no "tag" or special sequence/patch is present at all, the protein remains in the (soluble) cytoplasm.
B. The Big Divide -- Attach to the ER or not?
1. Ribosomes start to make protein first. Translation starts first; then ribosome location (attachment to ER or not) is determined by the sequence of the protein being made.
2. Which ribosomes go to ER: If protein has the right "tag" (a localization signal called a signal peptide (SP) or signal sequence) the ribosomes attach to the ER.
3. Which ribosomes stay in cytoplasm: If there is no SP, ribosomes remain "free" in the cytoplasm -- they do not attach to ER or any other membrane.
(Note: Becker says there are ribosomes translating mRNA in the nucleus. Maybe, maybe not. This finding is controversial -- the existence of nuclear ribosomes is not firmly established. So we are ignoring them.)
C. Fate of proteins made on free ribosomes
1. Soluble Cytoplasm -- the default location. If there are no "tags" at all, proteins stay in the cytoplasm.
2. Organelles. If proteins have the right "tags" they can be imported post-translationally (after synthesis) into organelles (nuclei, mito, chloro or peroxisomes) that are NOT part of the endomembrane system.
3. Terminology: "free" means not attached to a membrane. All ribosomes making protein, "free" or not, are attached to mRNA.
D. Fate of proteins made on attached ribosomes -- these become part of the endomembrane system and/or leave the cell.
1. They enter the ER by co-translational import (and some post-translational import, esp. in unicellular eukaryotes). Protein can
2. Most protein travel from ER to Golgi
(1). Suppose domain X of a multipass integral membrane protein is on the inside of the ER, sticking into the lumen of the ER. When a vesicle forms off the ER, where will domain X be? Inside vesicle in lumen? On outside of vesicle in cytoplasm?
(2). When vesicle fuses with Golgi, where will domain X be? In lumen of Golgi? Sticking out into cytoplasm?
3. Most proteins are sorted and processed in the Golgiand packed into vesicles that bud off the trans side of Golgi (also called TGN = trans Golgi network).
4. Where do the proteins and/or vesicles go next?
(vesicles involved in regulated secretion) → area near plasma membrane
a. Secretory vesicles
(1). Vesicles fuse with plasma membrane only in response to signal (such as hormone, change in ion concentrations, etc.)
(2). Fusion results in: Release of contents outside cell and/or addition of material to cell membrane. Click here for animation #1 -- annotated & animation #2 -- larger but not annotated.
b. Default vesicles (vesicles involved in constitutive secretion)→ plasma membrane → fuse automatically (constitutively) and release contents. Same as in (a) -- leads to addition of material to membrane or outside it. HOWEVER no signal is required for fusion. This is probably the "default" for proteins that are directed to the ER but have no additional directional information.
c.Vesicles containing hydrolases → Lysosomes (details to be discussed in future lectures).
d.Vesicles containing other enzymes → other parts of EMS (Some enzymes may stay in trans Golgi, but others bud off and go back to other parts of Golgi, ER, etc.)
Try problems 3-1, A & B, 3-13, & 3-16, A & B. You should be able to do 3-14, but you may have to look up the localization of some of the enzymes/proteins listed.
V. ER -- How does Co-translational ImportWork?
A. Signal hypothesis -- How ribosomes get to the ER & Protein enters ER -- Handout 6B. Steps below refer to handout. See Becker fig. 22-16 (20-16) or Purves 12.15
1. What is the Signal Hypothesis? Ribosome unattached to ER starts making protein. (step 1) If nascent (growing, incomplete) peptide has a "signal peptide," then ribosome plus growing chain will attach to ER membrane, and growing chain will enter ER as it grows.
2. How does ribosome get to the ER?
a. Signal peptide (SP) = section of growing peptide (usually on amino end) binds SRP. (step 2)
b. SRP = signal recognition particle
- Role: temporarily blocks translation; ferries nascent protein to ER.
- Structure: SRP contains proteins + RNA = Large particle containing both ribonucleic acid & protein like a ribosome or spliceosome.
c. SRP receptor on ER = docking protein. Binds SRP. (step 3). Translocon (gated pore or channel through membrane) is still closed.
3. How does growing chain enter the ER?
a. Ribosome/translocon complex formation occurs. (step 4)
- SRP is released, recycles -- GTP split
- Ribosome binds to pore/translocon
- Translocon opens & peptide enters (as loop).
- Ribosome resumes translation.
b. Translation (and movement through translocon) continues (step 5)
1. If protein goes all the way through the pore (as on handout) protein will end up as soluble protein inside the lumen of the ER.
2. If protein goes part way through, protein will end up as single pass protein in ER membrane. Details of how a transmembrane protein ends up inserted in the ER membrane will be explained next time for both single pass and multipass proteins.
c. Translation (and translocation) are completed, translocon closes. (step 6)
e. Signal Peptidase (usually) cuts off signal peptide on amino end at arrow. (step 7)
- Protein is released into lumen of ER
- Ribosome and mRNA are released
Now try problem 3-15.
B. (Next time): How do proteins get inserted into the ER membrane? Then, what happens inside the ER? The Golgi?