Plasma Membrane 1

Membrane Structure and Composition

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!

Wednesday 8/22/2012 10-12 p.m.

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/

Plasma Membrane 1 Learning Objectives

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.

Plasma Membrane 1 Learning Objectives (cont.)

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.

The Cell & Intracellular Organelles are Surrounded by a Membrane Bilayer

(A composite eukaryotic cell)

Plants

Source: Molecular Cell Biology, Lodish et. a. 4th ed, W.H. Freeman, 2000

Functions of the Plasma Membrane

Regulate nutrient & ion transport into the cell Regulate transport of waste out of the cell Maintain correct chemical conditions in the cell Provide a site for lipid-based chemical reactions Interact with other cells or the ECM Detect & transduce signals from environment to cell
Signal Transduction

Lipids & Many Membrane Proteins are Laterally Mobile in the Plane of the Membrane
The Fluid Mosaic Model

Plasma Membrane/Cell Membrane/ Cell Surface

Source: Addison Wesley Longman, Inc.

The Plasma Membrane - Topics Fluid Mosaic

The Basic Architecture of the Membrane
A.

1. Types of membrane lipids 2. Membrane proteins B. Membrane Fluidity C. Membrane Asymmetry

Source: Addison Wesley Longman, Inc.

Biological Membranes Have 2 Main Components- Lipids & Proteins

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

3 Common Types of Membrane Lipids 1. Phospholipids 2. Glycolipids 3. Cholesterol

Membrane lipids have an amphipathic nature.
2-loves the hydrophilic aqueous environment and the hydrophobic non-aqueous environment Hydrophilic (polar) head group Hydrophobic (non-polar) acyl side chains (tails)

Phospholipids
Basic Structure & Examples You do not need to know details of structure Why is it amphipathic?

Phospholipids are the Major Type of Membrane Lipid

1C A Phospholipid is Composed of 4 Groups 2C

16-18 C

3C

Hydrophobic

Hydrophilic
Source: Biochemistry, Stryer 5th ed. W.H. Freeman

Phosphatidate (Diacylglycerol 3-phosphate)

Example of a Simple Phosphoglyceride

Glycerol Backbone
Side chains

Phosphate head group at C3 of glycerol

C1 & C2

Amphipathic: Hydrophobic

Hydrophilic
Source: Biochemistry, Stryer 5th ed. W.H. Freeman

Commonly Occurring Membrane Phospholipids

(Phosphoglycerides)

Source: Biochemistry, Stryer 5th ed. W.H. Freeman

Membrane Lipids Can Also Include Carbohydrate Moieties

Glycolipids are sugar-containing lipids A sugar takes the place of the phosphate group

Sphingosine
Very enriched - where?
Source: Biochemistry, Stryer 5th ed. W.H. Freeman

Cholesterol is Common in the Plasma Membranes of Animals

4 Linked Hydrocarbon Rings

A lipid based on a steroid type structure

Hydrophilic

Hydrophobic
Source: Biochemistry, Stryer 5th ed. W.H. Freeman

In Aqueous Media Phospholipids and Glycolipids Readily Form a Bilayer Sheet

Polar Head Groups Groups Hydrophobic Interior Polar Head

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

Two Additional Structures Satisfy the 2-loves of Amphipathic Lipids

Micelles
Solubilization and purification of membrane proteins, LDL and bile particles are mixed micelles

Liposomes
* Used

in functional study of membrane proteins

TM

= membrane Or

protein * Important for drug delivery and therapeutics.

= drug
NOTE: Cut-away views half of a micelle and half of a liposome is shown

Why is this Bilayer Sheet Energetically Favored and Stable?

Membrane formation is due to the amphipathic nature of membrane lipids.

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.

The Plasma Membrane Fluid Mosaic

The Basic Architecture of the Membrane

A.

1. Types of membrane lipids 2. Membrane proteins B. Membrane Fluidity C. Membrane Asymmetry

Source: Addison Wesley Longman, Inc.

Lipid Bilayers Selectively Maintain Differences In ECF and ICF Concentration

Membrane
Category Examples
Simple diffusio n

The Concept of Selective Permeability

Some molecules can pass directly through lipid core. If they cannot, membrane proteins must serve as structures for passage. Channels, transporters, junctions, pumps.

Depends on cellular/organ setting

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

Proteins Carry Out Most Membrane Processes

Today, I will present basic concepts regarding membrane proteins. Next time, several specific membrane proteins will be discussed in detail.

Membrane Proteins are Typically Classified as Either Peripheral or Integral

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)

OUTER (LEAFLETS) INNER

Yellow = Integral Blue = Peripheral

EXOPLASMIC

CYTOPLASMIC

Source: Biochemistry, Stryer 5th ed. W.H. Freeman

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)

Often utilized by bacteria

Source: Biochemistry, Stryer 5th ed. W.H. Freeman

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

The membrane spanning -helix

A sequence of 20-25 AA, rich in hydrophobic/nonpolar AA with an -helical structure often serves as a transmembrane segment.

Why is the hydrophobic -helix perfectly suited to span the lipid core?

Example - Glycophorin Uses This Type of -Helix to Span the Membrane

The erythrocyte integral membrane glycoprotein glycophorin is the basis for the MN blood group
Hydrophobic AA are not restricted to membrane regions but a 20-25 AA stretch suggests a possible transmembrane domain. Hydrophobic or Neutral

(-) Charged (+) Charged

Sugar
(glycoprotein)
Source: Biochemistry, Stryer 5th ed. W.H. Freeman

Transmembrane -Helices Can Be Predicted from Hydropathy Plots

Hydrophobicity Index Transmembrane - helices may likely include: Alanine Valine Isoleucine Leucine Methionine Phenylalanine Tyrosine Tryptophan
Multiple -helices predicted:
+4 +3 +2 +1 0 -1 -2 -3

Inde x

1 1

2 2

3 3

4 4

Which AA are unlikely in a membrane-spanning region?

50

100

150

200
Source: Biochemistry, Stryer 5th ed. W.H. Freeman

Amino Acid

Schematic Representations - Membrane Proteins

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

* The hydrophobic side chains of this -helical

structure allow the protein to be tightly associated (integral) with plasma membrane
Source: Biochemistry, Stryer 5th ed. W.H. Freeman

Certain Proteins Rely on Specific Lipid Structures for Association with the Plasma Membrane
BOAT

Lipid Anchors are covalently attached to specific proteins.

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

Types of lipid anchors (structures) Farnesylation Palmitoylation Myristoylation GPI Anchor

(Glycosylphosphatidylinositol)
Source: Molecular Biology of the CellAlberts, et.al. 3rd ed.

Alterations and Defects in Membrane Proteins Underlie Many Human Diseases

Cancer: Alterations of membrane protein and/or lipid are
key to metastasis and invasion of tumor cells as they spread throughout the body.

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:

Reconstitution is Often Used to Study Function of Membrane Proteins Why?

Membrane proteins need to be present in a membrane bilayer, and with correct topology to function properly (even for lab studies)
They will denature otherwise

Source: Molecular Cell Biology, Lodish et. a. 4th ed, W.H. Freeman, 2000

Manipulation of Membrane Protein Function in Disease Treatment

Membrane proteins are highly useful drug targets:

PRILOSEC GASTRIC H+/K+ ATPase PROZAC Na+-coupled serotonin transporter Vast numbers of other membrane proteins . . .

The more details we know about membrane protein structure

and function the better we can: Make designer drugs targeting protein active sites Predict how mutations in a gene for a membrane protein may impact protein function In general, the more we know about a protein, the better we can treat disease

The Plasma Membrane Fluid Mosaic

The Basic Architecture of the Membrane
A.

1. Types of membrane lipids 2. Membrane proteins B. Membrane Fluidity C. Membrane Asymmetry

Source: Addison Wesley Longman, Inc.

Membrane Fluidity Why Is It Important?

Influences arrangement of proteins and lipids Foster assembly/disassembly of protein subunits and signaling complexes in the membrane Changes membrane permeability Excessive fluidity leads to membrane destruction Altering fluidity can alter membrane and/or cell function

Many Studies of Membrane Fluidity are Conducted in Experimental Settings Keep in Mind - Biological Membranes are Much More Complex !

Membrane Fluidity Effects of Fatty Acid Composition

Desaturated:

Kink disrupts tight packing of hydrophobic side chains

Saturated:
Allows tight packing of hydrophobic side chains
Source: Biochemistry, Stryer 5th ed. W.H. Freeman

A Distinct Melting Temperature (Tm) is Noted for Simple (Non-Biological) Membranes

Curve will shift left if: Curve will shift right if:

Shorter Acyl Chain Desaturated

Longer Acyl Chain Saturated

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

Tm: Phase Transition Temperature (a.k.a. Melting Temperature)

Source: Addison Wesley Longman, Inc.

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

Makes membranes less likely to

undergo phase transition

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

The Effects of Cholesterol on Membrane Fluidity Outcome: Maintains Proper Tm

Below Tm: Increases fluidity

* 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 Tm: Decreases 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

Membrane Lipids are Highly Mobile in the Plane of the Membrane

1 sec rapid rapid

very rare

rapid

rapid

L A T E R A L

Why is lipid flip-flop so very rare?

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

How Do We Know Fluidity of Membrane Proteins?

FRAP Method: F luorescence R ecovery A fter P hotobleaching
Note: Only 1 molecule of a membrane protein is shown, it is really in many on surface. Picture not drawn to scale.

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

FRAP Experimental Set up:

Cells (membrane protein) + Labeled Antibody ( ) =

Then Visualization

Recovered

Time
Recovered

Quantification

The Plasma Membrane Fluid Mosaic

The Basic Architecture of the Membrane
A.

1. Types of membrane lipids 2. Membrane proteins B. Membrane Fluidity C. Membrane Asymmetry

Source: Addison Wesley Longman, Inc.

Asymmetry is Key to Function of Membranes All Biological Membranes Are Asymmetric

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

Fatty acyl side chains also

show leaflet enrichment.

RBC: Cytoplasmic leaflet

enriched in unsaturated fatty acyl chains.

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.

Platelet activation and aggregation:

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.

Every Membrane Protein Has a Specific & Consistent Topology

Determined at time of their synthesis in the ER. Maintained during their journey to the (plasma) membrane. A protein requires proper topology for function. They dont flip flop

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 ?

Example: Blood Group Antigens

= glycosylation sites

Source: Source: Biochemistry, Stryer 5th ed. W.H. Freeman Molecular Biology of the Cell. Alberts. 3rd ed.1944 Garland

Each Cell Type Has a Unique Repertoire of

(Asymmetrically distributed)

Plasma Membrane Proteins

SDS-PAGE ANALYSIS

A. Erythrocytes B. Retinal rod cells C. Muscle (SR)

(Protein Gel Electrophoresis)

Separates proteins by size

Higher MW

S I Z E
MW

What is your interpretation of this SDS-PAGE?

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?*

Creation of RBC ghosts to study membrane function

SDS-PAGE analysis of RBC membrane proteins

Source: Molecular Cell Biology, Lodish et. a. 4th ed, W.H. Freeman, 2000

Major Proteins of the RBC Plasma Membrane

Integral: Glycophorins (A,B,C) single-pass, glycoprotein basis of MN blood group Anion exchange protein multi-pass exchange of choride for bicarbonate Also called band 3 Peripheral: Spectrin (, form dimer) Ankyrin Band 4.1 Actin G-3-P dehydrogenase Tropomyosin

Inward tension is created via cytoskeleton/membrane protein interaction, this determines cell shape and flexibility.

Proper Protein Interactions at the Membrane Are Key to Biconcave Shape/Function.

Clinical Example - Hereditary Spherocytosis

* 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?