C2006/F2402 '05 OUTLINE OF LECTURE #8

Handouts: 8A (Hand drawn; not on web.)

I. Wrap up of ER -- See notes of lecture #7 for details of I-III.

II. Golgi Complex -- Structure & Function

III. Transport Through the Golgi -- Are cisternae stationary or do they progress? See handout 8A, bottom.

IV. Lysosomes -- an example of sorting after the Golgi

A. 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? 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. 352-353 (358-359), if you are interested in more details.

B. Scavenger Pathway & Lysosomal diseases -- see Handout 8A, top.

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.)

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

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 hydrolase. The defect is in a gene for an enzyme that modifies the sugars attached to the 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 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.

V. Mitochondria & Chloroplasts How do proteins get into other cytoplasmic organelles that are not part of the EMS? See right fork 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 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. 20-19 (20-18).

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 8 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 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.

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 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.

VI. Peroxisomes -- summary of structure, function and synthesis. For more details see Becker pp. 358-362.

A. Structure -- comparison to mitochondria and lysosomes

1. Summary Table

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?

Biochemical Method: 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.

In situ Method: 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).

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 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.

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?

Peroxisomes have no DNA, and probably multiply by growth and division. (But see note below.)

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

All proteins of the peroxisome enter membranes post translationally. Most proteins go directly to peroxisomes from free ribosomes.

Localization signals for best known peroxisomal enzymes are on COOH end of protein. (Some perox. proteins have the signal elsewhere.)

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

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

Note: Some recent experiments raise the possibility that a few proteins destined for the peroxisomal membrane may enter the ER post translationally, associate with lipids in a a special area of the ER, and then go with the lipids to peroxisomes. The proteins and lipids from this area either add to pre-existing peroxisomes or give rise to new ones de novo (as vs. by splitting of pre-existing peroxisomes). However, all perox. proteins are made on free ribosomes, and most experiments favor the growth and division model.

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

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

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

B. Pore Function

1. Acts like (ungated, always open) channel for small molecules (may include small proteins).

2. 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. See Becker fig. 16-30 for a model.

What You Need -- Category	to Get into Nucleus	to Get into ER
Address/Localization Sequence	NLS	SP
Middle Man or "ferry protein"	Transporter proteins (importin)	SRP
Surface receptor protein(s)	Nuclear Pore Complex	Docking Protein (SRP receptor)

3. Transport is bidirectional -- mRNA, ribosomal subunits etc., must go out (see below) and proteins must go in. (See Becker fig. 16-29) How things get out is not as well understood as how things get in.

To review nuclear transport, try problem 4-2.

VIII. Summary of Transport, Localization signals, etc.

Issues to keep in mind -- (See bottom of handout 6A or 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 signal or sequence called?

What type of Transport?	Post-Translational			Co-translational
Which organelle is protein imported into?	Mitochondria	Peroxisomes	Nuclei	ER
Where on peptide is signal or localization sequence?	amino end	usually on carboxyl end	anywhere	usually on amino end^#
How many membranes crossed?	2*	1	?**	1
What is localization signal (or sequence/patch) called?	transit peptide (TP)	Peroxisomal LS	Nuclear LS (NLS)	signal peptide (SP)
Is signal removed?	yes	no	no	usually yes if on amino end

^#If protein is going to pass entirely into lumen of ER, SP is always on amino end. If protein is going to insert into the ER membrane, SP can be in middle and lock the protein into the membrane.

* This is for transport from cytoplasm into the matrix. Additional signals are needed to be retained in (or re-enter) the inner membrane, outer membrane, and/or intra-membrane space. Transport into chloroplasts is assumed to be similar.

** Depends how you count. Have to get across nuclear envelope consisting of two membranes, but passage is through pores and both membranes are crossed together. Answer could be 0, 1, or 2 depending on how you look at it.

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

IX. Nuclear Structure & Role of nucleolus -- Probably Next Time

A. Lamins A, B, C & Nuclear lamina

1. Lamins = IF's of nucleus (No MT or MF in nucleus)

2. Not to be confused with Laminin -- a glycoprotein in the extracellular matrix (ECM) found primarily in the basal lamina

3. Form nuclear cortex or lamina = web supporting the nuclear envelope -- see Becker fig. 16-31 (b). Similar function to ankryin/spectrin/actin web under plasma membrane.

4. State of Phosphorylation and function change during cell cycle

a. Phosphorylated at G2 --> M (prophase); de-polymerize; due to rise in protein kinase activity

b. Dephosphorylated at M --> G1 (anaphase); re-polymerize; due to rise in protein phosphatase activity

c. This is another example of using addition/removal of phosphate to control protein conformation & activity.

Kinases & phosphatases here are separate from their target proteins (as vs. case of Na⁺/K⁺ pump).

Same kinase or phosphatase can modify multiple target proteins at critical points in cell cycle.

5. May help organize chromatin; keep it proper distance from pores

B. Nucleolus

1. Nucleolus has no membrane (see Becker fig. 16-32).

2. Nucleolus is site of rRNA synthesis and assembly of ribosomal subunits -- production of ribosomes requires extensive traffic in and out of nucleus

a. rRNA is made in the nucleolus (see Becker fig. 16-33) using polymerase I (see below)

b. Ribosomal proteins are NOT made in the nucleolus.

Ribo proteins are made in the cytoplasm, but imported into the nucleus for assembly into subunits.

Messenger RNA for ribosomal proteins is made in the nucleus and exported to the cytoplasm, as for any other mRNA.

After the mRNA is translated, , the proteins are imported back into the nucleus for assembly with rRNA.

c. Ribosomal subunits are assembled in the nucleolus from ribosomal proteins (made in cytoplasm) and rRNA.

d. Completed ribosomal subunits are shipped out to the cytoplasm to be used in protein synthesis. Export out of the nucleus through the pores requires an NES (nuclear export signal). NES allows transport out of nucleus; NLS allows transport in to nucleus. Mechanism of transport through nuclear pores is thought to be similar, but not identical, in both directions.

3. Nucleolus (but not NOR, see below) disappears during meiosis/mitosis since genes for rRNA inactive then (no rRNA made or ribosomal subunits assembled)

4. Relationship of nucleolus to NOR (= nucleolar organizer region of DNA)

a. NOR = DNA coding for rRNA = rDNA = genes for rRNA. NOR is permanent -- is part of DNA.

b. Nucleolus forms at location of one or more NOR's. Several NOR's can cluster together to form one nucleolus. Nucleoli are only visible when NOR is transcribed and ribosomes are being assembled.

c. There are multiple copies of genes for rRNA; often many tandem copies per NOR. Multiple copies are needed because rRNA is made directly off the DNA -- no intermediate such as mRNA to allow for amplification. (Q to think about: Do you need multiple copies of the genes for tRNA?)

C. Multiple polymerases in euk. Differ in sensitivity to drugs, location in nucleus, & what RNA's they make. (See Becker table. 19-1 for details; for reference only.)

1. Types/Names of polymerases:

    a. RNA pol I --> big ribosomal RNAs. This is the polymerase found in the nucleolus.

    b. RNA pol II --> mRNA (We'll focus primarily on this one when we discuss regulation of transcription.)

    c. RNA pol III --> small RNA's like tRNA, 5S RNA (small ribosomal RNA)

2. How pol II works (and is regulated) -- how the genes to be transcribed are selected -- will be discussed at length below.

To review nuclear structure, try problem 4-5, parts A & C. Also look at 3-15, Questions A-C, for protein (5).

Next Time: Structure of Chromatin & Regulation of Gene Expression