C2006/F2402 '10 -- Outline Of Lecture #3 -- Last update 02/02/2010 03:11 PM

© 2010 Deborah Mowshowitz, Department of Biological Sciences, Columbia University, New York NY

Handouts:       3A -- Freeze Fracture;  Types of Membrane Proteins
                         3B --  RBC Membrane,  RBC -- Role of Anion Exchanger, & ECM

The video 'The inner life of the cell' -- short version with music -- is at http://www.studiodaily.com/main/searchlist/6850.html

The web site includes a web-sites page with links to web sites that you may find interesting and/or helpful. The web sites contain animations, explanations, pictures etc. that are relevant to this course.  (The list is not complete; I'll add to it as we go.) I will add specific links in the lectures, but you may want to explore some of the sites on your own. Please let me know if any of the web sites are useful, and/or if you find any other good ones.

Note: Minor changes are sometimes made to the notes after the lecture. (The time of the last update is always given at the start of the outline. All changes made after the lecture will be highlighted in blue  so you can find them easily. Anything significant will be noted on the corrections page.

I. Introduction to Membrane Structure 

    A. The Big Question: What does the structure seen in the EM represent?

    B. Lipid part

1. Amphipathic nature of lipids  -- See Sadava fig. 5.2  -- there are multiple different "two headed" lipids -- each type has a different structure, but each has a hydrophobic end and hydrophilic end.

2. Amphipathic Lipids form a bilayer.

    C. Protein part -- where are the proteins (relative to the lipid)? Is it a "unit membrane" or a "fluid mosaic?" 

For "unit membrane" See Becker fig. 7-4 for EM picture; for fluid mosaic model see Becker fig. 7-5 or Sadava 5.1.

1. Use of freeze fracture procedure 

a. E vs P faces of bilayer = surfaces you see if you crack bilayer open = inside of bilayer

(1). E face = inside of the monolayer that is closer to extracellular space (outside of cell)

(2). P face = inside of the monolayer that is closer to protoplasm (inside of cell)

b. What do you see on inside? See Becker fig. 7-16 & 7-17 or Sadava 5.3 or top panel on handout 3A. 

(1) Inside is not smooth -- shows proteins go through bilayer (implies "mosaic" model not unit membrane)

(2). More bumps (proteins) on P face than E face -- shows more proteins anchored on cytoplasmic (protoplasmic) side.

        2. Freeze fracture vs Freeze etch

a. Freeze fracture = crack frozen sample open, examine in EM;

b. Freeze etch = crack open, let some water sublime off to expose deeper layers, then look in EM. For some sample pictures, see Becker figs. 15-16, 15-19 (15-21),  15-25 (15-26), & 16-1.

    D. Fluid mosaic model -- overview of current idea of how proteins and lipids are arranged. See Becker fig. 7-5 (or 7-3) or Sadava 5.1. Also handout 3A, middle.

II. Fluid Mosaic Model of Membrane Structure  

    A. Fluid Part = Lipid bilayer

1. Formation of Bilayer  --  All amphipathic lipids form bilayers. In cell, virtually all lipids are inserted from one side of the bilayer (side facing the cytoplasm). How do lipids get distributed?

2. Lateral diffusion vs. flip-flop of lipids. See  Becker 7-10 & 7-11.

• lateral diffusion =  movement within plane of membrane -- fast (secs).   Animation of lateral diffusion.

• flip-flop = movement from one side of bilayer to the other -- slow (hrs) w/o enzymes. Enzymes (flipases = phospholipid translocators) are needed to speed flip-flop. (More details when we get to transport.)

3. Two sides of bilayer (leaflet) often have a different lipid composition.

    B. Mosaic Part = Protein. Types of Membrane Proteins -- what do you get if you take a membrane apart? See handout 3A, bottom panel.

1. Peripheral membrane proteins vs. integral membrane proteins

* A small number of integral proteins do not go all the way through the membrane; they will be largely ignored in this course. For examples see Becker fig. 7-19 (first protein on left) or Sadava 5.1 (last protein on right).

**Note that lipid-anchored proteins can be considered a type of integral protein or a separate category. See Becker fig. 7-19.

2. Transmembrane proteins of plasma membrane (See Sadava 5.4 and/or Becker fig. 7-19 & 7-21)

a. Single pass vs multipass 

b. Domains -- intracellular, extracellular, transmembrane 

c. Location of carbohydrates -- all in extracellular domain

d. Anchorage -- Some proteins are anchored to cytoskeleton; some float in lipid bilayer.

e. Types & Functions -- All bridge the membrane but function differs. Can be:

(1). Transport proteins -- Allow transport of small molecules

(2). Receptors -- Trap (bind) molecules on outside. Then receptor can:

    (a). Allow transport of trapped large molecules across membrane (by RME -- receptor mediated endocytosis)

    (b). Transmit signals to inside of cell from trapped molecule on outside of cell.

    (c). Do both -- transmit signal and internalize signal molecule.

(3). Connectors --  physically connect cytoskeleton (inside of cell) to materials on outside of cell (ECM = extracellular matrix) or to next cell.

Some transmembrane proteins act in more than one capacity.

III.  The Red Blood Cell (RBC) Membrane -- The best studied example of a Membrane. 

    A. Why RBC's

1. Easy to get

 2. No internal membranes  -- all organelles lost during maturation of human RBC -- see Becker fig. 7-20 (a). Only membrane = plasma membrane.

 3. Can make ghosts = resealed plasma membranes. Can be resealed (or broken and reformed into vesicles) in either orientation -- "right" or "wrong" side out.

    B. RBC membrane proteins -- Structure & Function. See Sadava fig.4-23 (6th ed.) or Becker fig. 7-20 (b) & 15-20 (15-22). (Handout 3B -- top)

1. Peripheral proteins -- spectrin, ankyrin, (band 4.1), actin.  Comprise peripheral cytoskeleton, which supports membrane. All cells are thought to have a similar structure under the plasma membrane.

2.  Intrinsic proteins -- Two basic kinds -- single pass & multipass.

a. Example of RBC Multipass  -- band 3/anion exchanger -- Catalyzes reversible exchange of the anions HCO₃^- (bicarb) and Cl^- between RBC and plasma. Exchange allows max. transport of CO₂ in blood (as bicarb in solution). See Sadava 48.14 or Becker 8-3.

(1). Why is transport of CO₂ an issue? Tissues carry out oxidative metabolism and generate lots of  CO_{2 .} The CO₂ diffuses out of the cells into the blood. However the solubility of CO₂ in plasma (cell-free liquid portion of the blood) is limited.

(2). Basic idea: Bicarb is much more soluble in plasma than CO₂, so lots of bicarb (but not much CO₂) can be carried in the blood. Therefore need to covert CO₂ to bicarb when want to carry CO₂ in blood; need to do reverse to eliminate the CO₂ (in lungs).

(3). Role of Carbonic anhydrase: Conversion of  CO₂ to bicarb (& vice versa) can only occur inside RBC, where the enzyme carbonic anhydrase is. (See handout 3B, middle panel.) Carbonic anhydrase catalyzes:

CO₂ +  H₂O ↔ HCO₃^- + H⁺

(4). Role of exchanger: Gases can pass through membranes by diffusion -- CO₂ can exit or enter RBC as needed. However bicarb cannot pass through membranes.  You need the anion exchanger to get bicarb in and out of RBC.

(5). Physiological Function of Exchanger

(a). Where CO₂ is high, as in tissues, CO₂ diffuses into RBC and is converted to bicarb inside the RBC. (Reaction above goes to right.) Then bicarb leaves RBC in exchange for chloride using the anion exchanger.

(b). In lungs,  the process is reversed --  bicarb reenters the RBC in exchange for chloride using the anion exchanger. The bicarb is converted back to CO₂ inside the RBC (reaction above goes to left). Then the CO₂ diffuses out of the cells and is exhaled.

(6). Note on structure -- in picture on handout, anion exchanger looks like a channel allowing simple diffusion of bicarb and Cl^- in and out.  Exchanger is actually more complex -- has moving parts and movement of each ion depends on movement of the other. More details on this & other types of transport proteins next time. 

b. Example of RBC single pass  -- glycophorin -- function of protein not known.

(1). Has large amount of (-) charged modified carbohydrate -- sialic acid. Possible functions:

(a).  Neg. charge may cause RBC to repel each other and prevent clumping of RBC.

(b). Loss of terminal sugars may occur with age and trigger destruction of "old" RBC.

(2). Glycophorins make up a gene family; variations in glycophorin A are  responsible for MN blood type differences. Variations in glycophorin C are correlated with resistance to malaria. 

    C. Proteins of Other Membranes -- Membranes of other cells are similar. In other membranes:

• Find proteins of same protein families as in RBC, as well as entirely different proteins.

• Find both intrinsic and peripheral proteins

• Intrinsic/integral proteins are both single and multipass proteins; anchored and floating. 

• Different membrane proteins are found in different cell types.

Try problems 1-2 & 1-3. To review membrane structure, try 1-15 to 1-17 & 1-20.

IV. Extracellular Matrix (ECM)

Note: Becker Chap. 17 goes well beyond what will be covered in this section. References to pictures and diagrams are included FYI. For a summary, see handout 3B, bottom. All these proteins are outside the cell. Made inside, and secreted (details later). A nice picture of the ECM is at http://219.221.200.61/ywwy/zbsw%28E%29/pic/ech4-1.jpg.

    A. Major Structural Proteins

• collagen  -- nice picture in Becker fig. 17-13 (17-2)

• elastin (diagram in Becker fig. 17-15 (17-4)).

    B. Adhesive Glyco-Proteins -- fibronectins, laminins, etc. Have multiple binding domains. Connect other materials in ECM with each other and/or connect to extracellular domains of transmembrane proteins. For pictures see Becker figs. 17-7 & 17-18 (17-6 & 17-7).

    C. Proteoglycans -- special type of glycoprotein consisting of lots of carbohydrate attached to a protein core. Provides a gel-like matrix for ECM. See Becker fig.  17-16 (17-5). Table below is for reference purposes only so you can follow the terminology. 
See http://themedicalbiochemistrypage.org/glycans.html for a nice web site with a summary of structure, function, and medical significance of proteoglycans and GAGs.