C2006/F2402 '04 -- Outline For Lecture #6
(c) 2004 Dr. Deborah Mowshowitz , Columbia University, New York, NY. Last update 02/05/2004 02:23 PM
Handouts: 6A: Protein Transport 6B -- Co-translational Import& Article from Science. (There is no 6C. You may have to use a Columbia computer to get the Science link to work.)
I. RME -- A few more examples -- Fe/Transferrin & EGF. See end of lecture #5 for details; bring handout 5B.
II. Labeling -- How do you follow material coming in (or going out) of the cell?
A. Types of Labeling
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, p. 831 and figs. A-19 & A-20. 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.
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.?
1. General idea -- Add labeled precursors, take samples after increasing time intervals, and measure radioactivity in dif. parts of the cell by autoradiography (or measurement of radioactivity in each 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 --> RER --> Golgi --> secretory vesicles --> outside (See Becker fig. 12-8.) Click here for animation.
Try problem 2-8 & 3-2D; by now you should be able to do all the problems in problem set 2.
III. Sorting of Proteins to their Proper Place: Overview. (See handout 6A & Becker fig. 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 usually called a localization sequence (if it consists of a continuous section in the peptide chain) or a patch (if it consists of a contiguous section in the folded protein).
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? Ribosomes making protein (translating mRNA) in the cytoplasm can become attached to ER or not. How do they "decide?" Depends on what protein they make. If protein has the right "tag" (a localization signal called a signal peptide or signal sequence) the ribosomes attach to the ER. Otherwise, ribosomes remain "free" in the cytoplasm -- they do not attach to ER or any other membrane. (Note: Becker 5th ed. 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.
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 possibly some post-translational import). Protein can
2. Most protein travel from ER to Golgi (a few may remain in ER) -- vesicles carrying protein bud off ER, travel to Golgi and fuse with cis side of Golgi (side nearest to nucleus).
(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 Golgi and packed into vesicles that bud off the trans sideof Golgi (also called TGN = trans Golgi network).
4. Where do the proteins and/or vesicles go next?
in response to signal (such as hormone, change in ion concentrations, etc.)
a. Secretory vesicles (vesicles involved in regulated secretion) --> area near plasma membrane
(1). Vesicles fuse with plasma membrane only
(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 below).
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 problem 3-1, A & B.
IV. ER -- How does Co-translational Import Work?
A. Signal hypothesis -- How ribosomes get to the ER & Protein enters ER -- Handout 6B. Steps below refer to handout. See Becker fig. 20-16 or Purves 12.15 (12.13)
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 protein + 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.
d. 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.
e. Translation (and movement through translocon) continues (step 5)
f. Translation (and translocation) are completed, translocon closes. (step 6)
g. Signal Peptidase (usually) cuts off signal peptide at arrow. (step 7)
- Protein is released into lumen of ER (Protein can also end up in the ER membrane as explained below.)
- Ribosome and mRNA are released
Now try problem 3-4, especially part D. (If some of the parts are not obvious, wait until later.)
B. How do proteins cross the ER membrane? (See handout 7A -- next time and fig. 20-17 of Becker)
1. How proteins enter/pass through the membrane -- important points
a. ER membrane contains gated pore (also called channel or translocon) for the growing chain.
b. SP probably forms loop not arrow. Loop enters channel (translocon) in membrane. SP loop is probably what opens (gates) the channel on the cytoplasmic side.
c. Growing protein chain passes through channel as chain is synthesized.
d. Where will protein end up? Protein can go all the way through the membrane and end up as a soluble protein in the lumen (as in example above) or protein can go part way through and end up as a transmembrane protein. Details below.
2. Types of Proteins that can result (see handout 7A)
a. Soluble protein in lumen. Happens if protein passes all the way through the membrane and SP (on amino end) is removed, as above.
b. Integral membrane protein anchored in membrane by SP with no cytoplasmic domain. This happens if SP is on the amino end and is not removed.
c. Single Pass transmembrane protein -- get one of 2 possibilities:
(1). Amino end on outside of cell (= on lumen side).
One way this could happen: If SP is on amino end, and SP removed, and there is a hydrophobic sequence (acting as a stop transfer sequence) in the middle of the peptide.
(2). Carboxyl end on outside.
One way this could happen: If SP is in the middle, not on amino end. SP in this case is not removed -- it becomes the transmembrane domain of the protein.
d. Multipass transmembrane protein. (Requires one SP and several hydrophobic (start/stop) sequences.
(1). Hydrophobic sequences can stop the process (of moving through pore) and anchor protein in membrane. (Protein slides out of pore, laterally, into lipid bilayer.) These are usually called "stop-transfer" sequences.
(2). Hydrophobic sequences in the middle of the peptide can restart looping --> multipass protein. These are usually called "start-transfer" sequences.
(3). Start and stop-transfer sequences are probably equivalent. Role depends on where in protein they occur.
(4). Every SP is also a "start transfer sequence" but not every start/stop sequence is necessarily an SP. To act as an SP, a sequence must bind to the SRP. A hydrophobic sequence that does not bind the SRP will not direct ribosomes to the ER.
e. Lipid Anchored Proteins: Proteins to be anchored to lipids on the outside of the plasma membrane are inserted into the ER. After the protein reaches the plasma membrane, the extracellular domain is detached from the rest of the protein and attached to lipid. (Proteins to be anchored to the plasma membrane on the inside are made on cytoplasmic ribosomes.) See Becker if you are curious about the details.
Now try problem 3-1C & 3-2 A -C. To test yourself on structure/insertion of integral membrane proteins, try 3-3.C. What happens inside ER
1. SP usually removed by signal peptidase (enzyme) inside ER.
2. Folding of protein -- requires chaperones.
a. Chaperones (also called chaperonins) are needed every time a protein remains unfolded or becomes unfolded to cross a membrane (or refolds on the other side). Two or more types of chaperones are involved in protein folding-- different ones are found in different parts of the cell.
b. Chaperones are of two major types (families) -- HSP 60 or HSP 70 depending on molecular weight (60 K vs 70 K) and mode of action. (See an advanced text if you are curious about the mechanisms.)
c. The major chaperone inside the ER is a member of the HSP 70 family, also called "BiP"
d. HSP = heat shock proteins = chaperones; these proteins were originally discovered because they are made in large amounts after exposure to high temperatures.
3. Make S-S bonds. In eukaryotes, all S-S bonds are formed in proteins inside the ER. Proteins made in the cytoplasm do not have S-S bonds. Cytoplasmic proteins do contain cysteines and have free SH groups.
4. Start N-glycosylation (oligosaccacharides are added to the N in the amide of asparagine side chains -- called N glycosylation.) See Becker fig. 12-7 if you are curious about the biochemical details.
5. Some proteins stay in ER (in lumen or membrane); most move on to Golgi.
D. What happens on outside of ER (besides protein synthesis)
1. Lipid synthesis -- lipids made and inserted on cytoplasmic side by enzymes attached to/in membrane. Lipids can reach parts of cell not connected to ER through vesicles and/or transport proteins.
2. Detoxifications and other reactions catalyzed by proteins on cyto side of ER. See text for details if interested.
To review the structure and function of the ER, try (or finish) problem 3-4.
Next Time: How do things get to and move through the Golgi? How do proteins get to other organelles?