Outline for Lecture 7

C2006/F2402 '04 OUTLINE OF LECTURE #7

Handouts: 7A = How proteins insert into ER membrane; 7B -- Golgi transport models & I-cell Disease (not on web)

I. What Happens in/on the ER, cont.

A. How proteins enter the ER membrane. (See IV, B of last lecture) & handout 7A.

To review how proteins enter membranes, try problems 3-1 & 3-3 (A).

B. 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) -- 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 ubiqutinalated 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.

A major proportion of all proteins made in cell (1/3?) do not fold properly and are degraded.

Destruction of many proteins is regulated -- level of protein activity can be controlled by protein degredation as well as by rate of synthesis, feed back of activity, modification, etc. More details and/or examples to follow.

C. 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.

II. Golgi Complex

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 & Traffic Flow - See Purves, fig 4.12 (4.17) 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 below.

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.

D. Sorting -- an example -- how proteins get to lysosomes (See Becker fig. 12-9) -- Normal Pathway

1. How are hydrolases identified & tagged for transport to lysosomes?

a. Localization Signal -- Most hydrolases destined for lysosomes have a special sequence/patch.

b. Reading the LS -- Enzyme(s) recognize the sequence/patch and add Mannose-6-P (N-glycosylation starts in ER by addition of standard oligosaccharide. Modification of standard sugars to M6P occurs in Golgi -- this modification only happens to proteins with the proper amino acid sequence = soluble hydrolases bound for lysosomes)

2. Role of M6P Receptor -- how is the tag recognized?

a. Binding the M6P -- Receptor in special part of trans Golgi binds proteins with M6P.

b. Sorting in trans Golgi -- Proteins with M6P and their receptors accumulate in coated pits and bud off --> sorting vesicle/endosome.

3. Sorting after the Golgi

a. Sorting vesicle/Endosome sorts multiple types of receptors and hydrolases. Same as what happens during RME when receptors and ligands are sorted.

b. M6P Receptors recycle back to Golgi; vesicles with hydrolases add to old lysosome or form new one.

c. SNAREs. How do various vesicles fuse when they reach the proper target? Complementary proteins on surface of target and surface of vesicle (SNAREs) match up. See SNARE hypothesis in Becker, p. 352-353 (358-359), if you are interested in more details.

E. Scavenger Pathway & Lysosomal diseases

1. I-cell disease -- what happens if the enzyme that catalyzes formation of M6P is missing.

a. The primary defect: In I-cell disease, the defect is in the gene for an enzyme that modifies all the soluble hydrolases. So many hydrolases, not just one, are affected. (Hydrolases that are membrane proteins are not affected.)

b. What happens to the hydrolases: All the soluble acid hydrolases lack M6P. So all the hydrolases that would normally stick to M6P receptors go to the wrong part of the Golgi. The hydrolases then end up in default vesicles. The default vesicles fuse with the plasma membrane and the hydrolases exit the cell. The hydrolases never reach the lysosomes.

c. The consequences: Inclusion bodies form = vesicles full of undigested materials that are normally degraded in lysosomes. ("Lysosomes" contain substrate, but no hydrolases to degrade the substrate.) Note that not all tissues are affected in I (inclusion) disease. This implies that there are other pathways for directing hydrolases to the lysosomes. Different enzymes and/or pathways must be critical in different tissues.

2. Standard lysosomal storage diseases -- what happens if one hydrolase is missing or defective. Examples: Gaucher's disease & Tay-Sach's disease. In these cases, only one hydrolase is missing due to a defect in the gene for that enzyme. (A different enzyme is missing in each disease.) All other hydrolases reach the lysosomes and function normally.

3. Salvage (scavenger) pathway -- recovers normal hydrolases that accidentally end up outside the cell.

a. Some M6P receptors are "misdirected" : they are not recycled to Golgi -- instead they reach plasma membrane in some cells (by default pathway?).

b. Some normal hydrolases (with M6P) are "misdirected" -- they reach extra-cellular fluid (by default pathway?) -- "escape" from cell.

c. Misplaced receptors can trap misplaced hydrolases: M6P receptors on the plasma membrane bind any extra-cellular hydrolases that "escaped" the cell (since these are normal hydrolases with M6P attached) . This is the "scavenger" part.

d. "Escaped" Hydrolases can be recovered by RME: Hydrolases bound to receptors are internalized by RME --> endosome --> lysosome = where they belonged in the first place. This is the "salvage" or recovery part. ("Misdirected" and "escaped" are in quotes above, because this may be a normal pathway that some hydrolases use to reach the lysosomes.)

4. Use of Salvage Pathway to treat Gaucher's Disease.

a. Missing lysosomal enzymes can be added: In cells with salvage pathway, added lysosomal hydrolases (containing M6P) can be taken up from outside. Hydrolases will be retrieved by RME and localized to lysosomes as explained above.

b. Practical Use: This method currently used to treat Gaucher's disease (one hydrolase missing) at annual cost of $50,000 per patient for added enzyme. Enzyme is so expensive because M6P addition (& glycosylation in general) cannot yet be done in bacteria. Enzyme must be obtained from eukaryotic cells grown in tissue culture -- in bottles.

c. Can't treat I-cell disease this way. Note only one enzyme is being replaced here, not an entire set of hydrolases (as would be required to treat I-cell disease).

To review lysosomes and lysosomal diseases, try problem 3-9.

III. 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.

IV. Post-Translational Import -- How do proteins get into other cytoplasmic organelles? See right fork of handout 6A.

A. How Proteins get into mitochondria & chloroplasts

1. Some proteins (not many) are made inside mitochondria and chloroplasts using organelle DNA and organelle transcription and translation machinery.

2. Most organelle proteins must be imported. Made on free ribosomes and then imported (post-translationally) into the organelles.

3. Organelle Membranes contain translocases. Proteins are imported by passing through pores or transport complexes (translocases) in the organelle membranes. See Becker 20-18.

4. Proteins can pass through both mito. membranes at once. Proteins enter matrix by crossing membranes at contact point = point where membranes are very close together and translocases of the inner and outer membrane are aligned. See Becker fig. 20-19 (20-18).

5. Localization signals. A short sequence called a transit peptide on the amino end is the usual localization signal that targets a protein to attach to a mito. translocase and enter the matrix. The TP is removed once the protein enters the matrix of the mitochondrion. (Additional signals are required to direct the protein to a membrane or the intramembrane space. See 8 below.) Similar sequences are used to direct chloroplast proteins to translocases on the organelle and to the correct organelle subcompartment.

6. Entry requires energy in form of ATP and/or electrochemical (proton) gradient. Note that transfer is post-translational so energy of protein synthesis cannot be used to drive entry of protein into organelle.

7. Chaperones are needed. Proteins are translocated in an unfolded state. Every time a protein remains/becomes unfolded to cross a membrane (or refolds on the other side) a chaperone is needed. Two or more types of chaperones involved here -- one in cytoplasm and one or two in matrix of organelle. See Becker fig. 20-20 (20-19).

a. Chaperones in cytoplasm keep protein unfolded or loosely folded until it reaches mitochondrion.

b. Chaperones inside organelle may help "pull protein in" and help protein fold properly once it enters.

c. Release of chaperones requires hydrolysis of ATP.

8. How do proteins reach parts of the organelle other than the matrix? Proteins destined for the membranes or the intramembrane space may enter the outer membrane, cross only part way in, and never reach the matrix -- the proteins could lodge in the appropriate membrane or the intramembrane space. Alternatively, the proteins may enter the matrix and then cross back out to the membranes or intramembrane space from the matrix. Different proteins probably use different pathways. In any case, proteins destined for sites other than the matrix require additional localization signals and/or stop/transfer sequences.

B. Peroxisomes (microbodies) and how proteins enter them

1. Comparison to lysosomes and/or mitochondria

Features like lysosomes	Features like mito/chloroplasts	Unique Features
Single Membrane	Probably grow and split	Signal (for import) on COOH end of proteins ; prob. not removed
No DNA	Protein enters post-translationally
Density & Size
Sequesters Dangerous Stuff		"Dangerous stuff" = product or substrate, not enzyme

2. How distinguished from lysosomes, since so similar in structure?

Separated from lysosomes biochemically ("grind and find" method) after growing animals on diet with triton (detergent) so lysosomes unusually light. Lysosomes accumulate detergent = mimic of phospholipid = amphipathic molecule with hydrophobic and hydrophilic ends. Density is proportional to protein/lipid ratio. Growth on detergent alters the ratio.

Identified in situ as different from lysosomes by marker enzymes = enzymes characteristic of and unique to a particular organelle. Typical marker enzymes are urate oxidase or catalase (for peroxisomes); acid phosphatase (for lysosomes).

3. Function = detoxification (in animal cells)

a. Oxidases and detoxification. Oxidases catalyze:

RH₂ + O₂ --> R + peroxide (H₂O₂)

Usually detoxifies RH₂ by increasing solubility (R generally more soluble and less hydrophobic than RH₂). Note that these reactions are real oxidations (involve actual addition of oxygen) not dehydrogenations (= removal of H's and electrons) as in most of energy metabolism. They generate peroxide, which is very reactive.

b. Catalase catalyzes:

H₂O₂ + R'H₂ (can be a second molecule of peroxide) ---> R' + 2 H₂O

If R'H₂ is a second molecule of peroxide, R' is oxygen and overall reaction is:

2 H₂O₂ --> O₂ + 2 H₂O

Catalase gets rid of peroxide and generates oxygen for another go round of oxidation or detoxifies R'H₂.

To review how proteins enter mitochondria and peroxisomes, try problems 3-6 & 3-7.

Next Time: Wrap up of transport into peroxisomes, and then on to Nuclei, Chromatin, & Regulation of Gene Expression