C2006/F2402 '08 OUTLINE OF LECTURE #8

(c) 2008 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/17/2008 01:47 PM

Handouts: 7C (Hand drawn; not on web) -- Golgi & Lysosomes

I. Golgi -- How does cargo (newly made proteins & lipids) move through the Golgi? Wrap up of the two models shown on Handout 7C, bottom. See last lecture for details.

II. Lysosomes -- an example of sorting after the Golgi  -- See Becker fig. 12-9 & Handout 7C, top.

    A. How proteins get to lysosomes -- Normal Pathway (steps refer to numbers on handout 7C)

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. 348-349 (352-353), if you are interested in more details.

    B. 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 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. (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.) 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

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 disease & Tay-Sach's 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 an enzyme that 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?). 

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 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. For a catalog of all lysosomal diseases & current treatment see eMedicine.

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 (20-18).

3. 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 (20-19).

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 intramembrane 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 (20-20).

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 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 (if they have a 'stop' transfer sequence) or stay in the intramembrane space. Alternatively, the proteins may enter the matrix and then use a 'start' sequence to cross back out to the membranes or intramembrane 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. 354-358 (358-362).

    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


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)

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'H2 is peroxide) or detoxifies R'H2 (by oxidizing it)

    C. How do Proteins (& phosopholipids) get into Peroxisomes?

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

V. How Proteins Enter Nuclei -- Nuclear Pores and Traffic through Them

    A. Pore structure -- hard to explain; will be demonstrated. See Becker fig. 18-27 & 28 (16-27 & 28) and/or Sadava fig. 4.8 (4.9)

    B. Pore Function

1. For relatively small molecules: Acts like (ungated, always open) channel for small molecules (may include small proteins). Transport is passive.

2. For  big  molecules: Acts as active transporter for big molecules. Features of active transport:

a. Transport requires energy (GTP split).

b. Requires NLS (nuclear localization signal) on protein transported into nucleus; signal not removed (may be needed again after mitosis to re-localize protein).

c. Involves binding to pore protein

d. Probably works like SRP/SP/Docking protein system with three components -- Protein with NLS binds to "middle man"  or "ferry proteins" (importins) which bind to pore. Additional proteins (that we will ignore) are required as well. See Becker fig. 18-30 (16-30) for a model.

What You Need -- Category

to Get into Nucleus

to Get into ER

Address/Localization Sequence



Middle Man or "ferry protein/particle"

Transporter proteins (importin)*


Surface receptor protein(s)

Nuclear Pore Complex

Docking Protein (SRP receptor)

*A middle man protein is required to enter or exit the nucleus, but different ones are used in for entry vs exit. Exportin is needed to get out of the nucleus, while importin is needed to get in.

3. Transport is bidirectional -- mRNA, ribosomal subunits etc., must go out (see below) and proteins must go in. (See Becker fig. 18-29 (16-29); molecular details are in 18-30.) How things get out is not as well understood as how things get in. Exit requires an exit signal (NES) and the transporter protein exportin.

To review nuclear transport, try problem 4-2.

VI. Summary of Transport, Localization signals, etc. (For Reference)

    A. FYI: Issues to keep in mind --  (See table below)

1. What type of localization signal (or localization sequence = LS) required? Where is it?

2. Is signal removed after membrane is crossed?

3. How many membranes must be crossed?

4. What is localization signal or sequence called?

5. When does translocation (crossing of membrane) occur -- during protein synthesis (co-translational) or after (post-translational)?

    B. Summary Table of Signal*** & Localization Sequences -- For Reference.

Post Translational Import Co-translational
Import Into: Mito/Chloro Peroxisomes Nuclei ER**
Signal: NH2 End * COOH end anywhere usually--NH2 end
Signal removed: Yes No No usually--yes
Enter or Cross: 2 membranes at contact point matrix 1 membrane pores 1 membrane
Name of Signal*** Transit Peptide (TP) Peroxisomal  Localization Signal Nuclear Localization Signal (NLS) Signal Peptide (SP)

*This is the signal needed to enter the matrix. Different and/or additional signals are needed to enter the outer membrane, inner membrane, or intramembrane space.

** Once protein enters ER, additional signals may be needed to direct protein to correct part of endomembrane system

*** The term "signal peptide" or "signal sequence" is sometimes used in a general sense to mean any of the localization signals listed here. It is used in a more specific way to mean only the localization signal used to enter the ER.

To review localization and transport, try problems 3-14 & 3-15.  By this point you should be able to do all the problems in Set 3 (except for protein (5) in 3-14 ).

Next Time: Structure of Nucleus; Structure of Chromatin & Regulation of Gene Expression.