C2006/F2402 '13 OUTLINE OF LECTURE #13
(c) 2010Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 03/04/2010 09:13 AM (Made a few formatting changes.)
Handouts: 13A &13B (hand drawn, not on web) and 13C.
13A -- Golgi transport
13B -- How hydrolytic enzymes get to Lysosomes
13C -- Tables comparing Mitochondria, Lysosomes, & Peroxisomes
I. What Happens in/on the ER?
A. What happens inside ER
1. Folding of protein -- requires chaperones. (For details, see notes of last lecture.)
2. 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. Requires signal peptidase (enzyme) and specific target sequence in substrate (newly made protein).
3. Some proteins stay in ER (in lumen or membrane); most move on to Golgi.
4. 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 (from ER or cytosol) 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. What comes out of the proteasome? Ubiquitin (recycled) and short peptide fragments of protein that was degraded.
f. 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 synthesiseins.
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 (exchange) prot
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.
II. Golgi Complex -- Structure & Function
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. 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. These oligosaccharides are 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.
Through the Golgi -- How does
cargo (newly made proteins) move through the Golgi? Are cisternae stationary or do they progress? See handout 13A.
1. What is known:
a. Transport vesicles
(1). Vesicles can bud off one sac (cisterna) of the Golgi and fuse with another in the same stack.
(2). In vitro, vesicles can fuse with cisternae of another stack. (See problem 3-10.)
b. Modification enzymes: Different modification enzymes are found in different parts of the Golgi (cis, medial trans). Same enzymes (marker enzymes) are always found in same part of Golgi.
c. Cargo: Cargo (newly made protein from the ER) moves through the Golgi from cis to trans.
2. Three big Issues (See table on 13A)
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. Overall: What happens to the composition of each sac? Does the content of modification enzymes change or the cargo?
B. Models -- see handout 13A.
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.
IV. Lysosomes -- an example of sorting after the Golgi -- See Becker fig. 12-9 & Handout 13B
A. What's in a lysosome?
1. Lysosomes contain two classes of protein.
a. Enzymes -- Many different acid hydrolases that digest different macromolecules.
b. Substrates of the hydrolases -- Proteins to be degraded.
2. All proteins to be degraded are enclosed in a membrane.
a. Most proteins to be degraded come from outside the cell by phagocytosis or endocytosis.
b. Some proteins to be degraded come from the cytoplasm, and are encircled by internal membranes. (Autophagy -- see texts if your are interested in more details.)
3. How do two classes of protein come together? Vesicle or compartment containing substrate (proteins to be degraded) fuses with vesicle containing enzymes -- either a vesicle with newly made hydrolases or a pre-existing lysosome.
B. How do hydrolases get to lysosomes? Normal Pathway (See top of handout 13B. Steps refer to numbers on handout.)
1. How do hydrolases reach the Golgi?2. How are hydrolases identified & tagged for transport to lysosomes?
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.
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).
c. Significance of this -- Many proteins are sorted in the Golgi, and the mechanism may be similar -- each class of cargo protein -- all those with a particular localization signal or tag -- may meet matching receptors in a particular part of the Golgi.
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.
C. Scavenger Pathway & Lysosomal diseases (See bottom of handout 13B)
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 13B.
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.
V. Peroxisomes -- summary of structure, function and synthesis. For more details see Becker pp. 356-360 (354-358). How do proteins reach organelles that are not part of the endomembrane system?
A. Structure -- comparison of peroxisomes and lysosomes
1. Summary Table -- See handout 13C. Features to consider:
1 Membrane(s) around organelle -- How many? 2 Contain DNA? 3 Grow & Split? 4 Where are organelle proteins made? Where are the ribosomes? 5 Protein import is co-translational or post-translational? 6 Localization Signal #1 =? 7 Additional LS? 8 Function of organelle?
2. 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 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 transported through blood** and 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.
** Cells that carry out oxidations generally have transporters to allow soluble material to exit cell and enter blood.
2. Role of Catalase: Catalase catalyzes:
H2O2 + R'H2 → R' + 2 H2O
R'H2 can be a second molecule of peroxide. In that case, R' is oxygen and overall reaction is:
2 H2O2 → O2 + 2 H2O
Catalase gets rid of peroxide, and
Catalase generates oxygen for another go round of oxidation (if R'H2 is 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.
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. If this happens, all the matrix proteins must be imported.
In summary: 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
peroxisomes, try problem 3-7.
VI. Mitochondria & Chloroplasts Where do their proteins come from?
A. Some Proteins are made inside the organelles
Some proteins (not many) are made inside mitochondria using organelle DNA and organelle transcription and translation machinery.
Mito make their own rRNAs & some or all tRNAs, but the rest of the transcription and translation machinery comes from outside.
Chloroplasts (in plant cells, in addition to mitochondria) have more DNA than mitochondria, and make more components inside than mito., but most chloroplast proteins are also imported from outside.
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 (TP) 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?
a. Additional localization signals and/or stop/transfer sequences are required -- in addition to a transit peptide. Different proteins probably use different localization signals and/or pathways.
b. One approach -- from the outside: 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 or hydrophobic anchor sequence) or stay in the intermembrane space.
c. An alternative approach -- from the matrix: Proteins destined for sites other than the matrix may enter the matrix first, and then use a hydrophobic 'start' sequence to cross back out to the membranes or intermembrane space from the matrix. (See problem 3-6.)
8. Summary Charts. See handout 13C for:
Comparison of Mitochondria vs Lysosomes & Peroxisomes
Comparison of Features of Peroxisomes to those of Mitochondria & Lysosomes
Next Time -- Wrap up of protein transport, and then: How do secreted signal molecules work at the molecular level? How do a few signal molecules produce a big effect in the target cell?