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Dr. Cynthia Smas cynthia.smas@utoledo.edu Dept. of Biochemistry & Cancer Biology Office:Rm 401D BHS; Lab: Rm 466 BHS 383-4527, 383-4131 Your questions are welcome at any time!
Plasma Membrane 1
References and Readings: Biochemistry (Stryer 7th ed.) Chapter 12 (relevant portions) Harpers Illustrated Biochemistry, 29th ed. Chapter 40.
Optional: Molecular & Cellular Biology (Lodish 4th ed, 2000.) Chapter 5 Available free on-line through NCBI/PubMed. http://www.ncbi.nlm.nih.gov/books/NBK21475/ (searchable only, cannot browse it)
Also note the 5th edition of Stryer is also free through NCBI/Books but is only searchable, not browsable http://www.ncbi.nlm.nih.gov/books/NBK21154/
1. Describe 3 common types of membrane lipids and discuss amphipathic nature of each. Indicate how membrane lipid structure facilitates self-assembly of the lipid bilayer. 2. Explain what types of molecules can pass directly through the lipid core of the membrane and which cannot and why. 3. Discuss the distinguishing features of integral and peripheral membrane proteins, using glycophorin as an example. 4. Explain how lipids can serve to anchor some proteins in the plasma membrane.
5. Explain the impact of fatty acid length and saturation, and the effects of cholesterol on membrane fluidity as reflected by the melting temperature (Tm). 6. Discuss one example of asymmetry of membrane lipids and of membrane proteins. For example, the impact of exoplasmic phosphatidylserine on cell destruction or the asymmetric nature of glycosylation of membrane proteins. 7. Discuss the types of interactions among the key protein components of the RBC membrane. Describe their contribution to its strength and flexibility in health & disease, for example in heriditary spherocytosis.
Plants
Source: Molecular Cell Biology, Lodish et. a. 4th ed, W.H. Freeman, 2000
Lipids & Many Membrane Proteins are Laterally Mobile in the Plane of the Membrane
The Fluid Mosaic Model
Lipids: Form a permeability barrier Define the basic architecture Proteins: Define the unique functions of membranes Determine selective permeability Transporters, channels, junctions Energy uptake, signal transduction
Protein to lipid ratio can vary with the cell type. This can be related to the function of that cell type.
Lipids
Water-insoluble biomolecules Highly soluble in organic solvents Great variety of structures Fuel and energy storage Signaling Membrane components
Phospholipids
Basic Structure & Examples You do not need to know details of structure Why is it amphipathic?
16-18 C
3C
Hydrophobic
Hydrophilic
Source: Biochemistry, Stryer 5th ed. W.H. Freeman
Glycerol Backbone
Side chains
C1 & C2
Amphipathic: Hydrophobic
Hydrophilic
Source: Biochemistry, Stryer 5th ed. W.H. Freeman
Sphingosine
Very enriched - where?
Source: Biochemistry, Stryer 5th ed. W.H. Freeman
Hydrophilic
Hydrophobic
Source: Biochemistry, Stryer 5th ed. W.H. Freeman
Cross Section
Two faces of the plasma membrane (leaflets): Exoplasmic - toward the extracellular environment Cytoplasmic - toward the intracellular environment
Source: Molecular Cell Biology, Lodish et. a. 4th ed, W.H. Freeman, 2000
Liposomes
* Used
= membrane Or
= drug
NOTE: Cut-away views half of a micelle and half of a liposome is shown
Hydrophobic interactions by exclusion of water drives formation of energetically stable structures. Van der Waals attractive forces between hydrocarbon tails favor close packing. Electrostatic and H-bond attractions between the polar head groups and water molecules also involved.
In summary:
The lipid bilayer membrane is a cooperative structure that is formed & maintained by multiple noncovalent interactions. The role of lipids in the plasma membrane are to define the basic bilayer architecture which acts to form a permeability barrier for the cell.
Some molecules can pass directly through lipid core. If they cannot, membrane proteins must serve as structures for passage. Channels, transporters, junctions, pumps.
Source: Molecular Cell Biology, Lodish et. a. 4th ed, W.H. Freeman, 2000
The PROTEINS of the membrane determine its functional complexity for each specific cell type.
Define the unique functions of membranes Determine selective permeability Examples - transporters, channels, junctions Function in energy uptake, signal transduction
Peripheral - More loosely associated with the membrane emoved with mild conditions (salt/pH) Do not enter or span the hydrophobic core Integral More tightly associated with the membrane Removed only with harsh conditions (detergents). Enters or spans the hydrophobic core
(Single-pass or multiple-pass)
EXOPLASMIC
CYTOPLASMIC
Specific Secondary Structures Enable Polar Proteins to Happily Exist in the Hydrophobic Lipid Core
-Helix Most commonly used in higher organisms Barrel (A single -helix is circled in blue)
Membrane Proteins Often Have an -Helical Structure to Span the Lipid Bilayer
The peptide bond is polar Proteins contain charged AA But the membrane core is hydrophobic! An -helix can function to span the core
Why is the hydrophobic -helix perfectly suited to span the lipid core?
Inde x
1 1
2 2
3 3
4 4
50
100
150
200
Source: Biochemistry, Stryer 5th ed. W.H. Freeman
Amino Acid
Source: Biochemistry, Stryer 5th ed. W.H. Freeman; Source: Addison Wesley Longman, Inc.
Prostaglandin H Synthase: An Example of an Integral* Membrane Protein That Enters but Does Not Span the Membrane
Certain Proteins Rely on Specific Lipid Structures for Association with the Plasma Membrane
BOAT
ANCHOR
Protein
Thy-1,Ras, Src, or others
Lipid anchors are hydrophobic and can embed in the hydrophobic core of the plasma membrane, to anchor (localize) the protein at the membrane.
DOCK Lipid Core ANCHORS of the Plasma Membrane
Diabetes:
Defective insulin signaling, defective function of glucose transporters (More in next lecture) Defective cell-cell communication (example connexins in arrhythmias, will be discussed by other instructors)
Heart Disease:
Source: Molecular Cell Biology, Lodish et. a. 4th ed, W.H. Freeman, 2000
PRILOSEC GASTRIC H+/K+ ATPase PROZAC Na+-coupled serotonin transporter Vast numbers of other membrane proteins . . .
Many Studies of Membrane Fluidity are Conducted in Experimental Settings Keep in Mind - Biological Membranes are Much More Complex !
Desaturated:
Saturated:
Allows tight packing of hydrophobic side chains
Source: Biochemistry, Stryer 5th ed. W.H. Freeman
Membrane Fluidity
Phase transition
More fluid
Tm lower Need less heat energy to disrupt the membrane Tm higher Need more heat energy to disrupt the membrane
Less fluid
Fatty Acyl Chain Length Effect on Membrane Transition Temperature (Melting Temperature)
* *
Regulation of membrane fluidity by alteration of fatty acyl chain length and saturation is employed by bacteria.
Source: Biochemistry, Stryer 5th ed. W.H. Freeman
Cholesterol is the Key Determinant of Membrane Fluidity in Animals Disrupts regular interactions
of fatty acyl side chains
Cholesterol acts as a buffer against changes to membrane fluidity Cell membranes need to have a correct degree of fluidity for function. This requires the right amount of cholesterol in the cell membrane.
Source: Molecular Biology of the Cell. Alberts, et.al. 3rd ed.1944 Garland
* Below the melting temperature, the membrane is gel-like or more solid * The lipid side chains are tightly packed and orderly * Introducing a kinked structure disrupts this, increases fluidity
* Above the melting temperature, the membrane is more fluid-like * The lipid side chains are disorganized and moving * Cholesterol acts to limit/restrict the overall free movement of the lipid side chains due to its planar shape (steroid nucleus), thus making it less fluid
Source: Molecular Biology of the Cell. Alberts, et.al. 3rd ed.1944 Garland
very rare
rapid
rapid
L A T E R A L
Membrane Proteins Also Have Lateral Mobility The size of the molecule This depends on: Interactions with other molecules Temperature Lipid composition of the membrane The composition of the protein
Done on live cells -in real time. Uses an antibody specific for the membrane protein you want to study. (Can also use other types of tools such eGFP fusion proteins . . .) The antibody also has a fluorescent label linked to it (to allow us to see if).
Location of the protein under study shown by fluorescence (green) signal (fluorescence microscopy used). Signal in any area of membrane can be obliterated (bleached by a laser). The recovery of signal in bleached area (green) for your protein is observed. This measurement shows rate of movement of this protein in the membrane.
Source: Biochemistry, Stryer 5th ed. W.H. Freeman
Then Visualization
Recovered
Time
Recovered
Quantification
The exoplasmic and cytoplasmic surfaces have different proteins and different enzymatic and other activities. An asymmetric distribution occurs for both membrane lipids & proteins. This asymmetry is key to proper function. What are some examples of this asymmetry for membrane lipids & proteins?
Membrane Asymmetry - Each Leaflet of the Membrane Has A Distinct Lipid Composition The choline -containing phospholipids are mostly exoplasmic. The amino-phospholipids are mostly cytoplasmic. Flippases, floppases & scramblases can impact lipid
asymmetry.
Choline-containing
Amino-containing
Altered Distribution of Membrane Lipids Can Target Cells for Destruction Exposure of
phosphatidyl serine on the exoplasmic leaflet occurs in physiological and pathologic states.
Cascade of thrombin activation and protein C pathways Activation of alternative complement pathways
Recognition and removal of cells: RBCs: Exposed phosphatidyl serine causes macrophage recognition and destruction of these RBCs in the spleen.
Apoptosis (programmed cell death): Cells undergoing apoptosis expose phosphatidyl serine on their exoplasmic leaflet. This triggers macrophage recognition and destruction of apoptotic cells.
Cytosolic
Exoplasmic
Source: Molecular Biology of the Cell. Alberts, et.al. 3rd ed.1944 Garland
Glycosylation Sites are One Example of Asymmetry for Some Membrane Proteins
Some membrane proteins are glycosylated, occurs at sites in their exoplasmic portion. Complex sugar groups added in ER & Golgi by successive reactions.
(many types, much variety )
Can be N-linked (asparagine) or O- linked (serine, threonine). Occurs only in the lumen of ER & Golgi. Can confer specificity & function.
Remember Glycophorin ?
Source: Source: Biochemistry, Stryer 5th ed. W.H. Freeman Molecular Biology of the Cell. Alberts. 3rd ed.1944 Garland
Higher MW
S I Z E
MW
Lower MW
Source: Biochemistry, Stryer 5th ed. W.H. Freeman
A Variety of Membrane Protein Interactions Gives Strength & Flexibility to the Fluid Mosaic
Source: Molecular Cell Biology, Lodish et. a. 4th ed, W.H. Freeman, 2000
The RBC is a Key Example of the Relationship Between Membrane Proteins and Disease
Biconcave shape facilitates gas exchange. Flexible shape for travel through tight spots (capillaries). The functionality of the RBC is closely tied to plasma membrane integrity. Defects in RBC membrane proteins are often indicated by clearly visible alterations in RBC morphology.
The Red Blood Cell Plasma Membrane is One of the Most Thoroughly Studied
*Why?*
Inward tension is created via cytoskeleton/membrane protein interaction, this determines cell shape and flexibility.
* Mutations in genes for spectrin, ankyrin (or other genes) leads to ... * Weakened interaction of peripheral and integral membrane proteins * Cytoskeletal architecture altered * Detected by osmotic fragility test * Autosomal dominant (1/5000). * Spherocytic cells subject to destruction in the spleen - anemia.
Which blood smear is normal RBC morphology & which is abnormal (H.S.), why?
Due to defects in some RBC membrane proteins, H. S. RBCs lack flexibility and clog up splenic sinusoids, & are destroyed.
Key Concepts
The plasma membrane is a selective permeability barrier that is essential for proper cell function. Proteins and amphipathic lipids are the two main components of the bilayer membrane. Membranes form spontaneously and are stable. The lipids of the membrane define the basic architecture. The proteins impart specific functions. The lipids and proteins of the plasma membrane are and dynamic (fluid) and the asymmetrically distributed. Plasma membrane proteins can interact with extracellular signals (hormones, other cells, the extracellular matrix, other) and the cytoskeleton.
In my next lecture: How does the plasma membrane function in communication between cell and environment?