C2006/F2402 '05 OUTLINE OF LECTURE #7

(c) 2005 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/07/2005 07:03 PM

Handouts: 6A from last time; 7A -- Signal Hypothesis -- Co-translational Import ;  7B =  How proteins insert into ER membrane

I. Sorting of Proteins to their Proper Place: Overview, cont. (See handout 6A & Becker fig. 20-14 or Purves 12.14 -- terminology in Purves is slightly different.) 

   A. What do you call the amino acid sequence that determines the fate (final location) of each individual 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 start to make protein.  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).

Questions:
(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?

a. Secretory vesicles (vesicles involved in regulated secretion) --> area near plasma membrane

(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 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.

 II. ER -- How does Co-translational Import Work?

    A. Signal hypothesis -- How ribosomes get to the ER & Protein enters ER -- Handout 7A. Steps below refer to handout. See Becker fig. 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 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)

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)

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 7B 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 7B)

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 finish problem 3-1 & try 3-2, A -C. To test yourself on structure/insertion of integral membrane proteins, try problem 3-3.

III. What Else Happens in/on the ER?

    A. What happens inside ER 

1. Terminal SP usually removed by signal peptidase (enzyme) inside ER.

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). 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.

6. What happens to proteins in ER that do not fold properly? See Becker p. 733-734.

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 small protein called ubiquitin. (See Becker or advanced texts if you are interested in the enzymatic details.)

c. Degraded by Proteasome -- proteasome = a large protein complex in cytosol that degrades ubiquitylated proteins  to fragments, at expense of ATP. Major site of degradation of cellular proteins.

d. What goes to the proteasome? Proteins that are misfolded, damaged, or have served their function.

    B. 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 (exchange) 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 problem 3-4.

IV. Golgi Complex -- Structure & Function

    A. How things get there -- from ER in coated vesicles (coat made of protein called coatomer or COP instead of clathrin). For animation of how materials pass from ER to and through Golgi click here.

    B. Structure & Overall Traffic Flow -  See Purves, fig 4.12  or Becker fig. 12-4 & 12-8.

1. Two sides  -- to review:

a.  cis/forming face (side closest to nucleus & ER)

b. trans/maturing face (away from nucleus)

2. Three basic parts or compartments

a. CGN (cis-Golgi network) or cis Golgi

b. medial cisternae (sacs) -- part in between 'cis' and 'trans' Golgi

c. TGN (trans-Golgi network) or trans Golgi

2. 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.

3. Sacs in stack connected by vesicle traffic  -- not completely clear which way transport vesicles go or what they carry.  It is clear that newly made protein and lipid passes through the Golgi from the cis face to the trans face, and that vesicles from one stack can fuse with another in vitro. Models for transport through the Golgi are  discussed in more detail later. (If no time today, we'll discuss them next time.)

    C. Function -- what reactions take place inside Golgi?

1. Finish N glycosylation  --  oligosacch. that was added to glycoproteins in ER is modified. Oligosacch. is attached to "N" of amide side chains of asparagines (asn's). 

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.

V. Transport Through the Golgi -- Are cisternae stationary or do they progress?

    A. Background

1. Transport vesicles can bud off one sac (cisterna) of the Golgi and fuse with another. However it is not completely clear which way the transport vesicles go or what they carry. 

a. Direction of vesicle traffic: is it cis to trans = forward = anterograde, or vice versa = retrograde? (Older models assumed it was forward, but current evidence indicates it is both -- some vesicles go in one direction and some the other, as shown in Becker fig. 12-8.)

b. What do the vesicles carry: Is it newly made proteins from the ER and/or enzymes to modify the new proteins?

2. Net Direction of Transport. It is clear that newly made protein and lipid pass through the Golgi from the cis face to the trans face.

3. Vesicles from one stack can fuse with cisternae of another stack in vitro. (See problem 3-10.) 

    B. Models

1. "Vesicle Transport Model" or Stationary Cisternae Model

a. Transport vesicles move primarily forward (towards trans face).

b. Vesicles carry newly made proteins (from the ER) to next part of Golgi for additional modifications. 

c. Sacs of Golgi (with their characteristic enzymes) stay put, and newly made proteins pass from sac to sac by means of vesicles. 

d. Enzyme composition of each sac stays the same. Each part stays in the same place and holds on to its characteristic enzymes. It is the substrates of the enzymes (the newly made proteins) that pass through, carried by the vesicles from sac to sac. 

2. "Cisternal Maturation Model"  = a modified Progression Model 

a. Transport vesicles move primarily retrograde (towards cis face).

b. Vesicles carry enzymes to modify and sort proteins. They do not carry the newly made proteins from the ER. 

c. Sacs of Golgi move, carrying newly made proteins & lipids inside. New sacs are constantly formed at the cis face from material transported from the ER. Old sacs are lost from the trans face as they age.  

d. Enzyme composition of each sac changes with time. The enzyme composition of each individual sac is constantly changing as it ages and passes from cis to trans face. However the characteristic enzymes found in the sacs at each position of the Golgi (cis, medial & trans) remain the same, because the enzymes are "passed back." The vesicles retrieve the enzymes of "older" sacs (closer to the trans face)  and carry them back to newer sacs (closer to the cis face). 

3. Connection Model -- another possibility is that the Golgi sacs are actually connected (although they seem to be separate), and that proteins move back and forth  from one sac to the next, although net bulk flow of newly made proteins is from cis to trans. Some recent data using genetically modified protein tagged with GFP has been interpreted in support of this model.

4. What really happens? The models above are not mutually exclusive, so a hybrid model is possible. Only new experiments generating new data will settle the question of how material actually moves.  Not all materials may move through the Golgi in the same way.

To review traffic through the Golgi, try problem 3-10.

Next Time: Sorting in the TGN: How do normal hydrolases get to lysosomes?  What happens if (a) one hydrolase is not made or (b) all the hydrolases lack M6P? How do enzymes get to peroxisomes?