C2006/F2402 '09 OUTLINE OF LECTURE #7
(c) 2009 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/09/2009 05:07 PM
Handouts: 7A: Protein Transport 7B -- Signal Hypothesis -- Co-translational Import & 7C = How proteins insert into ER membrane. (You also need 6A.)
nice video (& explanation) of a pulse chase experiment, try this link:
This site has many interesting animations. To select a topic, go to http://sumanasinc.com/webcontent/animation.html
I. Labeling -- How do you follow
(newly made) material going out of the cell?
See last lecture for details of A & B.
A. Types of Labeling (using added tracers)-- continuous, or pulse-chase. B. Detection -- How do you find where the radioactivity (or whatever tracer/label you used) is?
2. Fractionate First
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 discussed last time, emphasis was on following molecules going in to the cell. This example is about following newly made molecules on their way out.
-- Add labeled precursors (small molecules) and measure incorporation into macromolecules.
1. General idea
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 measure amounts in each isolated fraction.
2. A specific example-- following secreted proteins out. See handout 6A.
a. Continuous label vs pulse-chase results (See handout 6A & fig. 12-10 of Becker. 6th ed. has curves; 7th ed. has autoradiographs.)
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 of newly made material, try 3-4D.
D. Another Type of Labeling -- Cell makes its own labeled (fluorescent) protein containing GFP
GFP has been mentioned before. Here are the details.
GFP = green fluorescent protein = small fluorescent protein made by jelly fish. (Click here for page with pictures of GFP and related fluorescent proteins.) GFP is used as tag to follow proteins inside the cell. GFP is not added from outside. Instead, genetic engineering is used to splice the gene for GFP to gene for protein of interest. The recombinant gene makes a fusion protein = normal sequence of amino acid + sequence of amino acids in 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.
For examples of use of GFP, see Becker fig. A-14, or Purves p. 885 (7th ed). Not in 8th ed. (Sadava).
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 article.
See also: http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/presentation-speech.html.
See problem 2R-4 for an example of the use of GFP labeling.
II. Sorting of Proteins to their Proper Place: Overview (See handout 7A & Becker fig. 22-14 (20-14) or Sadava 12.15 (12.14) -- terminology in Sadava is slightly different.)
A. What determines the fate (final location) of each individual protein? The amino acid sequence of the protein itself. The ability of each protein to reach its proper destination is built into the protein. The presence (or absence) of localization signals in the amino acid sequence of the protein 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. (See bottom of handout 7A for a summary.)
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 for a soluble protein. 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
- Remain inserted in membrane -- become a transmembrane single or multipass protein (details below)
- Pass all the way across the membrane and end up in lumen of ER (as soluble protein or peripheral, membrane associated protein on lumen side)
2. Most proteins 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 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 local Na+ concentration, etc.)
(2). The 'signal' usually causes an increase in intracellular Ca++, which directly triggers the fusion, causing exocytosis.
(3). 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, 3-16, A & B, & 3-17. You should be able to do 3-14, but you may have to look up the localization of some of the enzymes/proteins listed.
III. ER -- How does Co-translational Import Work?
A. Signal hypothesis -- How ribosomes get to the ER & Protein enters ER -- See handout 7B. Steps listed below refer to handout. See Becker fig. 22-16 or Sadava fig. 12.16 (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) does not bind directly to the ER. Binds to 'middleman' called SRP. (Step 2.)
b. SRP = signal recognition particle = example of an RNP (ribonucleoprotein 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.
- Helps attach ribosome to ER, so growing chain can enter pore (translocon) in ER.
3. How does Growing Chain enter ER? Takes two steps (3 & 4)
a. ER has SRP receptor. Receptor is also called docking protein. SRP binds to SRP receptor/docking protein, not directly to pore. (Step 3.)
b. ER has Translocon (gated pore or channel through membrane) which allows growing chain to pass through membrane as chain is made. Pore is closed until ribosome with growing chain gets into position.
Note on terminology: The term "translocon" is used in (at least) 2 different ways. Sometimes it is used to mean only the channel itself, and sometimes it is used to mean the whole complex of proteins required for translocation of proteins across the membrane -- the channel, SRP receptor, etc. It should be clear from context which usage is meant at any one time.
c. Ribosome/translocon complex formation occurs. (step 4 = complex step involving several events)
- SRP is released, recycles -- GTP split
- Ribosome binds to pore/translocon
- Translocon opens & peptide enters (as loop).
- Ribosome resumes translation.
4. How does a new protein end up in the lumen?
. Translation (and movement through translocon) continues (step 5)
b. Translation (and translocation) are completed, translocon closes.(step 6)
c. Signal Peptidase cuts off signal peptide at arrow.(step 7)
Now try problem 3-13, especially part D. (If some of the parts are not obvious, wait until later.)
B. How do proteins cross or enter the ER membrane? (See handout 7C and/or fig. 22-17 of Becker)
1. How proteins enter/pass through the membrane -- important points
a. 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.
b. Protein enters as it is made. In humans, growing protein chains usually enter the ER as the chains are synthesized (co-translational import).
Note: In unicellular organisms, proteins often enter the ER after they are finished (post-translational import). Post translational import into the ER will be ignored here, but is covered at length in cell biology.
c. How do transmembrane proteins get anchored in the membrane? A hydrophobic sequence may trigger opening of the pore sideways, so protein slides out of pore, laterally, into lipid bilayer. These hydrophobic sequences are called 'stop-transfer' sequences and/or 'anchor' sequences.
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, on 7B) or protein can go part way through and end up as a transmembrane protein. Depends on sequence of protein.
2. Types of Proteins that can result (see handout 7C)
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). Type 1: Amino end is on lumen side of membrane (on E side); Carboxyl end is in cytoplasm (on P side of membrane)
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). Type 2: Carboxyl end is on lumen side of membrane (on E side); Amino end is in cytoplasm (on P side)
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. (SP doubles as stop-transfer sequence.)
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, as explained above.
(2). Hydrophobic sequences in the middle of the peptide can restart looping → multipass protein. These are usually called "start-transfer" sequences (see 4).
(3). 'Start-transfer' and 'stop-transfer' sequences are probably equivalent. Role depends on where in protein they occur. (Both start- and stop-transfer sequences are also called 'topogenic sequences' as they determine the topology of the finished peptide.)
(4) A sequence that starts or restarts passage of a protein through the translocon is usually called a 'start transfer sequence' even if it also doubles as a stop.
(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. If a hydrophobic sequence does not bind the SRP, it 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 generally made as follows: Protein is made on RER and inserted into the ER membrane. 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 finish problem 3-1 & 3-4, A -C. To test yourself on structure/insertion of integral membrane proteins, try problems 3-2 & 3-3.
IV. What Else Happens in/on the ER?
A. What happens inside ER
1. Terminal SP usually removed. Signal peptidase (enzyme) inside ER recognizes a particular sequence of amino acids next to the SP. If this sequence is present in the protein, signal peptidase cuts the peptide chain at that point. (If this sequence is absent, SP is not removed.)
2. Folding of protein -- requires chaperones.
a. Chaperones (also called chaperonins) -- proteins needed to assist in protein folding. Chaperones are used every time a protein remains unfolded or becomes unfolded to cross a membrane (or refolds on the other side). Different chaperones are found in different parts of the cell.
b. Chaperones are of two major types (families) -- HSP 60 (forms barrel) or HSP 70 (binds to hydrophobic regions). Differ in 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. Why are chaperones named HSP 60, HSP 70? HSP = heat shock protein. Chaperones, aka HSP's, are made in large amounts after exposure to high temperatures. (That's how they were first discovered.)
3. Enzymatic Modifications. The appropriate enzymes inside the ER catalyze the following:. 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.
a. Making of S-S bonds
b. Start of N-glycosylation . Oligosaccacharides are added to the N of the amide of asparagine side chains (this is called N glycosylation.) See Becker fig. 12-7 if you are curious about the biochemical details. Additional steps of glycosylation occur in the Golgi; details below.
c. Removal of SP as above.
4. Some proteins stay in ER (in lumen or membrane); most move on to Golgi.
5. What happens to proteins in ER that do not fold properly? See Becker p. 750-752 (755-757) .
a. Transport to cytosol -- Unfolded proteins are transported back to the cytosol (through the translocon -- mechanism unknown).
b. Ubiquitin addition -- in cytosol, proteins to be degraded are marked for destruction by addition of a multiple molecules of a small protein called ubiquitin to side chains of lysine. (See Becker or advanced texts if you are interested in the enzymatic details.)
c. Role of Proteasome = a large protein complex in cytosol that degrades ubiquitinylated proteins to fragments, at expense of ATP. Major site of degradation of intracellular proteins. (Proteins from outside are generally degraded in lysosomes.)
d. What goes to the proteasome? Proteins that are misfolded, damaged, or have served their function.
A major proportion of all proteins made in cell do not fold properly and are degraded.
Destruction of many proteins is regulated -- level of protein activity can be controlled by protein degradation as well as by rate of synthesis, feed back of activity, modification, etc. More details and/or examples to follow.
e. 2004 Nobel Prize for Chemistry was awarded to Aaron Ciechanover, Avram Hershko and Irwin Rose for the discovery of the ubiquitin/proteasome system.
B. What happens on outside of ER (besides protein synthesis)
1. Lipid synthesis --(exchange) proteins.
a. Insertion: Lipids made and inserted on cytoplasmic side (cytoplasmic leaflet) of membrane by enzymes attached to/in membrane.
b. Flipping: Enzymes ('flippases' = transporters) are required to move amphipathic lipids from one leaflet (P side) of membrane to other leaflet (E side). If lipids are moved preferentially from one side of membrane to the other, transport is active and requires ATP.
c. Transport: Lipids can reach parts of cell not connected to ER through vesicles and/or transport
2. Some detoxifications and other reactions are catalyzed by proteins on the cyto side of ER. See text for details if interested.
To review the structure and function of the ER, try problem 3-4.
1. Two sides of stack
a. cis/forming face (side closest to nucleus & ER)
b. trans/maturing face (away from nucleus)
2. Three basic parts or compartments in a stack
a. CGN (cis-Golgi network) or cis Golgi -- may include fusing vesicles
b. medial cisternae (sacs) -- part in between 'cis' and 'trans' Golgi
c. TGN (trans-Golgi network) or trans Golgi -- may include budding vesicles
3. Different marker enzymes/functions found in different parts. (See Becker figs 12-5 & 12-6) Enzymes unique to any one cell organelle or compartment are called 'marker enzymes' = their presence is a 'marker' for the presence of that compartment or organelle.
4. Sacs in stack connected by vesicle traffic -- not completely clear which way transport vesicles go or what they carry. (See below.) It is clear that newly made protein and lipid passes through the Golgi from the cis face to the trans face, as shown on this animation.
C. Function -- what reactions take place inside Golgi?
-- oligosaccharide that was added to glycoproteins in ER is modified. Oligosacch. is attached to "N" of amide side chains of asparagines (asn's).
1. Finish N glycosylation
2. Do O glycosylation of glycoproteins. Sugars are added to "O" of the hydroxyl of the side chain of ser & thr.
3. Assemble sugars of proteoglycans (linear chains of repeating sequence = GAGs)
4. Concentrate, sort proteins. This occurs at trans face (TGN). Different areas of Golgi have receptors that trap proteins going to different destinations.
To review how proteins are directed to the right place and modified in the ER and Golgi, try problem 3-2.
Next time: How are materials transported through the Golgi stacks?