C2006/F2402 '09 OUTLINE OF LECTURE #8

Handouts: 8A & 8B (Hand drawn; not on web). 8A -- Golgi transport models; 8B -- How hydrolytic enzymes get to Lysosomes

I. Transport Through the Golgi -- How does cargo (newly made proteins) move through the Golgi? Are cisternae stationary or do they progress? See handout 8A.

A. Background

1. What is known: Transport vesicles can bud off one sac (cisterna) of the Golgi and fuse with another in the same stack. In vitro, vesicles can fuse with cisternae of another stack. (See problem 3-10.)

2. Three big Issues (See table on 8A)

a. Direction of vesicle traffic: Which way do the vesicles go?

1. cis to trans = forward = anterograde?

2. trans to cis = backward = retrograde?

3. Both? 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?

1. Cargo proteins -- Newly made proteins from the ER, and/or

2. Modification Enzymes -- Used in the Golgi to modify the newly made cargo proteins

c. Do cisternae move? What carries the newly made material from cis to trans, the cisternae or the vesicles?

d. Consequences: What happens to the composition of each sac? Does the content of modification enzymes change or the cargo?

B. Models -- see handout 8A.

1. "Vesicle Transport Model" or Stationary Cisternae Model

a. Transport vesicles move primarily forward (anterograde) -- towards trans face. For an animation of this process, see here.

b. Vesicles carry cargo -- vesicles carry newly made proteins from one part of Golgi to next part for additional modifications.

c. Sacs of Golgi (& their characteristic enzymes) stay put -- newly made proteins (cargo) pass from sac to sac by means of vesicles.

d. Net Result: Enzyme composition of each sac stays the same. Each part stays in the same place and holds on to its characteristic ('marker') enzymes. It is the substrates of the enzymes (the newly made cargo 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. Net Result: 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 (FYI) -- 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. There isn't much evidence or enthusiasm for this model, but 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. For a review of the models and the data as of 1998 (the 100th anniversary of the discovery of the Golgi) see Coming to Grips with the Golgi.

To review traffic through the Golgi, try problem 3-10. See also problems 3R-8 & 3R-9.

II. Lysosomes -- an example of sorting after the Golgi -- See Becker fig. 12-9 & Handout 8B

A. How proteins get to lysosomes -- Normal Pathway (See top of handout 8B. Steps refer to numbers on handout.)

1. How do hydrolases reach the Golgi?

a. Enzyme synthesis: Hydrolases are made on ribosomes bound to ER. Enzymes enter ER co-translationally (as they are made).

b. Transport to Golgi: Hydrolases are transported in vesicles to Golgi. Steps 1 & 2.

2. 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) Step 3.

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

a. Binding the M6P -- Receptor in special part of trans Golgi binds proteins with M6P. Step 4.

b. Sorting in trans Golgi -- Proteins with M6P and their receptors accumulate in coated pits (step 5) and bud off (step 6) and go to a sorting vesicle/endosome (step 7).

4. 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. (step 7)

b. Recycling: M6P Receptors recycle back to Golgi (steps 8A & 10); vesicles with hydrolases add to old lysosome or form new one (steps 8B & 9). Note that 8A & 8B are equivalent to the same numbers on the handout of RME. 8B goes to the lysosome and 8A recycles back to the membrane from which it came.

c. SNAREs. How do various vesicles fuse when they reach the proper target? There are matching transmembrane proteins (SNAREs) on the target membrane and the vesicle membrane. The cytoplasmic domains of the proteins are complementary, and pair up with each other. See SNARE hypothesis in Becker, p. 351-352 (348-349), if you are interested in more details.

B. Scavenger Pathway & Lysosomal diseases (See bottom of handout 8B)

1. I-cell disease (ICD) -- 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 are affected, not just one. (Hydrolases that are membrane proteins are not affected.) Step 3 is skipped.

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. (as in Step 11) The default vesicles fuse with the plasma membrane and the hydrolases exit the cell. (Step 12) 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.)

(1). Not all lysosomal enzymes are affected in I (inclusion) disease. This implies that there are other pathways for directing hydrolases to the lysosomes. (See problems 3-18 & 3R-1 for examples of alternative pathways.)

(2). Not all tissues are affected in I cell disease. For example, the liver is not affected in ICD. This implies that in different tissues, either different pathways (to lysosomes) are used, and/or that different enzymes must be critical for proper lysosomal function.

2. Standard lysosomal storage diseases

a. What is a lysosomal storage disease? It's what happens if one hydrolase is missing or defective. A different enzyme is missing in each disease.

b. Examples: Gaucher's or Gaucher (pronounced 'Go-shay') disease & Tay-Sachs disease. In these cases, only one hydrolase is missing due to a defect in the gene for that enzyme. All other hydrolases reach the lysosomes and function normally.

c. How is I disease different? In I disease, most of the hydrolases are missing from lysosomes -- the hydrolases are made, but are not transported to lysosomes. The defect is not in the gene for a particular hydrolase. The defect is in a gene for a modification enzyme. This enzyme modifies the sugars attached to many different hydrolases.

d. Genetics: Each standard lysosomal storage disease or I disease is caused by a single, recessive mutation -- but the mutation in each disease is different.

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?). See dashed line in bottom of handout 8B.

b. Some normal hydrolases (with M6P) are "misdirected" -- they reach extra-cellular fluid (by default pathway?) -- "escape" from cell. (As in steps 11 and 12.)

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 always use to reach the lysosomes.)

4. Use of Salvage Pathway to treat Gaucher's Disease -- Enzyme Replacement Therapy.

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 is 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. Several other lysosomal diseases have been treated by enzyme replacement therapy at a similar cost per patient.

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. See also 3-18 & 3R-1. For a catalog of all lysosomal diseases & current treatment see eMedicine. There are many additional articles in eMedicine about genetic & metabolic diseases.

III. Mitochondria & Chloroplasts How do proteins get into other cytoplasmic organelles that are not part of the EMS? See right fork of flow chart on top of handout 6A.

A. Some Proteins are made inside the organelles

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

B. Most organelle proteins must be imported.

1. Most organelle proteins are made on free ribosomes and then imported (post-translationally) into the organelles.

2. Organelle Membranes contain translocases. Proteins are imported by passing through pores or transport complexes (translocases) in the organelle membranes. See Becker 22-18.

3. How to reach the matrix? 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. 22-19.

4. 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 intermembrane space. See 7 below.) Similar sequences are used to direct chloroplast proteins to translocases on the organelle and to the correct organelle subcompartment.

5. 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.

6. Chaperones (chaperonins) 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. 22-20.

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.

7. How do proteins reach parts of the organelle other than the matrix? Proteins destined for the membranes or the intermembrane space may enter the outer membrane, cross only part way in, and never reach the matrix -- the proteins could lodge in the appropriate membrane (if they have a 'stop' transfer sequence) or stay in the intermembrane space. Alternatively, the proteins may enter the matrix and then use a 'start' sequence to cross back out to the membranes or intermembrane space from the matrix. (See problem 3-6.) 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.

8. Comparison of Mitochondria vs Lysosomes

	Feature	Lysosomes	Mitochondria
1	Membrane(s) around organelle	Single	Double
2	Contain DNA?	No	Yes
3	Grow & Split?	No	Yes
4	Where are organelle proteins made? Where are the ribosomes?	On Rough ER	Free in Cytoplasm & inside organelle
5	Protein import is	co-translational	post-translational (while unfolded)
6	Localization Signal #1	signal peptide	transit peptide
7	Additional LS	signal to add M6P	stop transfer &/or others (depends on final location)
8	Function of organelle	Degradation of macromolecules	Energy Metabolism

IV. Peroxisomes -- summary of structure, function and synthesis. For more details see Becker pp. 356-360 (354-358).

A. Structure -- comparison to mitochondria and lysosomes (see table above)

1. Summary Table -- compare to table above

	Feature	Peroxisomes
1	Membrane(s) around organelle	Single
2	Contain DNA?	No
3	Grow & Split?	Yes; may also arise de novo
4	Where are organelle proteins made? Where are the ribosomes?	On Free Cytoplasmic Ribosomes
5	Protein import is	post-translational (while folded)
6	Localization Signal #1	on COOH end; probably not removed
7	Additional LS	None?
8	Function of organelle	Detoxification; fatty acid oxidation (in animals)

2. Another way to Compare Features to those of Mito. & Lysosomes

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 made in cytoplasm on free ribosomes.	Protein crosses membrane in folded state.
Density & Size	Protein enters post-translationally
Sequesters Dangerous Stuff	Some energy metabolism (fatty acid oxidation)	"Dangerous stuff" = product or substrate, not enzyme

3. How are peroxisomes distinguished from lysosomes, since both organelles are so similar in structure?

a. Biochemical Method: Peroxisomes separated from lysosomes biochemically ("grind and find" method) after growing animals on diet with triton (detergent). Details:

Lysosomes accumulate detergent.

Detergent = mimic of phospholipid = amphipathic molecule with hydrophobic and hydrophilic ends.

Density of organelles is proportional to protein/lipid ratio. Growth on detergent alters the ratio in lysosomes.

Lysosomes with triton (equivalent to extra lipid) are unusually light (low density, because of high lipid/detergent content).

Density of peroxisomes is unchanged by growth on detergent.

b. In situ Method: Peroxisomes identified in situ as different from lysosomes by marker enzymes

Marker enzymes = enzymes characteristic of and unique to a particular organelle.

Typical marker enzymes for peroxixomes are urate oxidase (function unknown) or catalase

Typical marker enzyme for lysosomes is acid phosphatase

B. Major Function = detoxification (in animal cells). See Becker for other roles, esp. in other organisms.

1. Role of Oxidases: Oxidases catalyze:

RH₂ + O₂ → R + peroxide (H₂O₂)

Oxidation detoxifies RH₂ by decreasing toxicity, and/or by increasing solubility

R is generally more soluble and less hydrophobic than RH₂. Soluble material is more easily excreted by the kidneys; hydrophobic material is more likely to accumulate in the body (& reach toxic levels).

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.

These reactions generate peroxide, which is very reactive.

2. Role of Catalase: 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

Net Result:

Catalase gets rid of peroxide, and

Catalase generates oxygen for another go round of oxidation (if R'H₂is peroxide) or detoxifies R'H₂ (by oxidizing it)

C. How do Proteins (& phosopholipids) get into Peroxisomes? Here is a summary of the details:

1. Matrix Proteins

Peroxisomes are not considered part of the endomembrane system -- all the proteins of the matrix are made on cytoplasmic free ribosomes.

All proteins of the peroxisomal matrix enter the organelles post translationally. Matrix proteins go directly to peroxisomes from free ribosomes.

Localization signals for best known peroxisomal matrix enzymes are on COOH end of protein. (Some perox. proteins have the signal elsewhere; not all signals are the same. See problem 3-19, esp. part C.)

Mechanism of import is not well understood -- some matrix proteins enter without unfolding. Entry requires ATP.

2. Membrane lipids

Some phospholipids of peroxisomal membrane are made on the ER. Carried to peroxisome by transport/exchange proteins and/or vesicles.

Some lipids are made in the organelle.

3. Membrane Proteins

The proteins of the membrane and the proteins of the matrix enter the organelle by separate pathways.

Some membrane proteins may be made on the ER, but this is not settled.

Assembly of peroxisomal membranes may involve a special area of the ER. A few proteins destined for the peroxisomal membrane may be made on the RER, associate with lipids in this special area of the ER, and then go with the lipids to pre-existing peroxisomes (via vesicles).

4. How do new peroxisomes form?

Peroxisomes have no DNA, and have been seen to multiply by growth and division.

Under certain circumstances, vesicles from the ER are able to give rise to new (empty) peroxisomes de novo as well as adding to pre-existing peroxisomes as explained above. (All the matrix proteins must be imported in any case.)

All perox. matrix proteins are made on free ribosomes, and most experiments favor the growth and division model as the main source of new peroxisomes.

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

Next Time -- How do proteins enter nuclei? How do ribosomal subunits get out of the nucleus?