1. The Nucleus Nuclear Organization Nuclear Envelope and Molecular Trafficking Nucleolus and rRNA Processing
The nucleus is one of the main features that distinguishes eukaryotic cells from prokaryotic cells.
-You might even say that the nucleus serves as the regulatory center or the cell.
-It serves several function including;
-storage of DNA
-site of DNA synthesis, transcription and RNA processing – gene expression is regulated by post-transcriptional modifications in the nucleus. HOW?
-transport by way of nuclear envelope
-processing of rRNA

2. The Structure and Function of the Nuclear Envelope
The nuclear envelope has a complex structure consisting of two nuclear membranes, an underlying nuclear lamina, and nuclear pore complexes.
Nuclear membranes are a system of two concentric membranes (inner and outer) that surround the nucleus.
The nuclear envelope separates the contents of the nucleus from the cytoplasm and provides the structural framework of the nucleus.
The nuclear envelope separates the contents of the nucleus from the cytoplasm and creates a structural framework of the nucleus and acts as a barrier that prevents the free passage of molecules between the nucleus and the cytoplasm. This helps to maintain the integrity of the nucleus as a distinct biochemical compartment.
-It consists of 2 nuclear membranes, a lamina and nuclear pore complexes.

3. 9.1 The nuclear envelope
The selective traffic of proteins and RNAs through the nuclear pore complexes establish the internal composition of the nucleus and also play a critical role in regulating eukaryotic gene expression.
cell4e-fig jpg In this electromicrograph you can see the dual membrane that surrounds the nucleus. You can also see a nuclear pore complex and how the membrane of the endoplasmic reticulum integrates with the nuclear membrane. Both of these things become important when we talk about transport.
-The selective traffic of proteins and RNAs through the nuclear pore complexes helps to establish the composition of the nucleus. It also serves to regulate gene expression in eukaryotic cells.
-The nuclear pore serves to transport molecules into and out of the nucleus and the ER serves to process and transport protein to the cell surface.

4. Structure of the Nuclear Envelope
The critical function of the nuclear membranes is to act as a barrier that separates the contents of the nucleus from the cytoplasm.
The nuclear lamina underlies the inner nuclear membrane and is a fibrous meshwork that provides structural support to the nucleus.
Lamins are 60- to 80- kilodalton fibrous proteins that make up the nuclear lamina.
Let’s talk about the specifics of the structure of the nuclear membrane. As I mentioned before, it consists of 2 nuclear membranes, nuclear pore complexes and an underlying nuclear lamina.
-The nucleus is surrounded by 2 concentric membranes called the inner and outer membranes. The outer membrane is integrated with the endoplasmic reticulum which allows the space btw. The inner and outer membrane is connected with the lumen of the ER. This membrane is similar in structure to the ER membrane. It has ribosomes on its cytoplasmic surface.
-The inner membrane carries only proteins that are need inside the nucleus…..for example proteins that bind to the nuclear lamina.
-The nuclear lamina is the structure located on the inside surface of the inner nuclear membrane. It is a fibrous network that serves to create the nuclear structure This fibrous network is composed of kDalton fibrous proteins called lamins. Groups of these lamins form the fibers you see on the inside surface of the nuclear membrane.

5. Structure of the Nuclear Envelope
Lamins are composed of intermediate filament proteins which combine with each other to form higher-order structures.
Attachment of Lamins to the nuclear envelope is mediated by prenylation and binding to specific inner nuclear membrane proteins such as emerin and the lamin B receptor.
Nuclear Lamina diseases: Hutchinson-Gilford progeria
-Lamins are composed of intermediate filament proteins….these proteins also serve to maintain the structure of the cytoskelaton.
-Most mamallian cells have 3 lamin genes, A, B and C, that code for 7 different lamin proteins. These 7 lamin proteins combine with each other to form the structure of the lamina.
-How do they form the complex structure of the lamina? Two lamin proteins interact forming a dimer. Two alpha helical regions of 2 polypeptide chains join together forming these coiled coils. Polymers of these dimers make up the nuclear lamina. Often this occurs by the head to tail association of dimers which leads to formation of linear polymers and side by side association of polymers -
-How do these lamins associate with the inner nuclear membrane? This attachment is mediated by the posttranslational addition of lipid (prenylation of C-terminal residues) on the lamins. Also, Lamins bind to specific inner nuclear membrane proteins such as emerin and the lamin B receptor, mediating their attachment to the nuclear envelope and localizing and organizing them within the nucleus.
-The lamina also attaches to chromatin by way of histones H2A and H2B.
-The nuclear lamina really creates a mesh network throughout the nucleus…..many of the proteins necessary for DNA replication, transcription and RNA processing bind to the lamina. So in essence the lamina serves to facillitate many of the events that occur in the nucleus.
-What happens if there are mutations in one of the lamin genes? Defects in production of the lamin A protein results in the disease Hutchinson-Guilford progeria.

6. Nuclear Lamina Defects
X-linked Emery-Dreifuss muscular Dystrophy
Results form mutations in the nuclear envelope protein, emerin.
Non-Sex Linked Emery Dreifuss Muscular Dystrophy
Results from mutations in the LMNA gene which is a single gene that encodes lamin A and C.
Hutchinson-Guilford Progeria Disease
Results from a 150 bp deletion within the lamin A gene.
-Both of the Emery Dreifuss Muscular Dystrophies are characaterized by stiff knees, elbows, neck and heels and electrical defects in the heart. Generally these symptoms occur by age 10. By age 20, it progresses to serious heart defects which may require a pacemaker. These progresses to gradual wasting and weakness of the upper arm and lower leg muscles.
-Hutchinson-Guilford Progeria disease is characterized by premature aging.
-It was originally thought that these sorts of nuclear lamina defects would cause generalized structural defects in rapidly dividing cells. This does not seem to be the case. There appears to be a tissue specific expression pattern of these mutated proteins. It is not clear why this is the case.

7. The Nuclear Pore Complex
A nuclear pore complex consists of a structure with eightfold symmetry organized around a large central channel.
Nuclear pore complexes are the only channels that are present in the nuclear membrane to allow small polar molecules , ions and macromolecules (proteins and RNAs) which permit the transport from the nucleus to the cytoplasm.
-These pore complexes consist of eight structural subunits that surround a central channel. It is a very large structure with a mass of about 125 million daltons. For comparative sake, it is about 30 larger than a ribosome.
-In vertebrates, the nuclear pore complex consists of different pore proteins called nucleoporins.
-The nuclear pore complex plays a very important role in the physiology of cells….it regulates what gets into and out of the nucleus of a cell.

8. The Nuclear Pore Complex
Nuclear pore complexes are the only channels through which small polar molecules, ions, and macromolecules can travel between the nucleus and the cytoplasm.
Depending on their size, molecules can travel through the nuclear pore complex by one of two different mechanisms.
Passive transport
Active transport –energy dependent
The structure of the nuclear pore complex: There are 8 spokes arranged around a central channel. The spokes are connected to rings at the nuclear and cytoplasmic surfaces. The spoke ring assembly is anchored within the nuclear envelope at sites of fusion between the inner and ourter nuclear membranes. Protein filaments extend from the cytoplasmic and nuclear rings, forming a distinct basketlike structure on the nuclear side.
The movement of molecules into and out of the nucleus is critical to normal eukaryotic cell function.
-RNA is synthesized in the nucleus and must be exported to the cytoplasm for protein synthesis.
-Also, proteins that perform nuclear functions such as transcription factors must be transported into the nucleus.
-The nuclear pore complex allows proteins to be continuously shuttled between the nucleus and cytoplasm.
-The size of the molecule that must be transported into or out of the nucleus, one of two mechanism is typically utilized:
-If the molecules are less than kDaltons in size, then they pass freely through the pore in either direction. These molecules diffuse freely through open aqueous channels (9 nm wide) in the pore complex. This type of transport is called passive transport.
-Most proteins and RNAs are too large to be transported through these open channels. These macromolecules pass through the nuclear pore complex by an active or energy dependent means of transport. They are selected transported across the nuclear membrane through the use of energy.

9. Selective Transport of Proteins to and from the Nucleus
Nuclear export signals are specific amino acid sequences that target proteins for export from the nucleus.
Protein import through the nuclear pore complex begins when a specific importin binds to the nuclear localization signal of a cargo protein in the cytoplasm.
How are proteins transported across the nuclear membrane?
-Selective transport of proteins to and from the nucleus is generally mediated by proteins or portions of protien. Many protiens contain nuclear localization and nuclear export signals or sequences that are needed to allow entry into or out of the nucleus.
- In addtion there are protiens such as importins and exportins that functions to shuttle molecules into and out of the nucleus.

10. Selective Transport of Proteins to and from the Nucleus
Nuclear localization signals are specific amino acid sequences that are recognized by transport receptors and direct the transport of proteins through the nuclear pore complex.
Nuclear localization signals have been identified in many proteins.
There are lots of proteins that are essential to the normal functioning of the cell. Since all proteins are synthesized in the cytoplasm by the ribosome, many of these proteins must be transported to the nucleus inorder to drive processes such as transcription and replication. These are protiens such as DNA polymerases, RNA polymerases, histones and transcription factors.
-How do they get into the nucleus? They enter the nucleus by way of the nuclear pore complex and are targeted to the nuclear pore complex by a nuclear localization signal that is present on most nuclear proteins. They have specific amino acid sequences called NLS that serve to send the proteins into the nucleus. These sequences are recognized by transport receptors that transport the proteins through the nuclear pore complex.
-There have been numerous localization signals identified to date. The first nuclear localization signal was identified in the simian 40 (SV40) T antigen which is a virus encoded protein that initiates viral DNA replication in infected cells. It is 7 AA in length.
-Sometimes the localization signals are short stretches of basic amino acids called classic NLS and in other cases the NLS consists of amino acids that are close together, but not adjacent to each other(called bipartite NLS).

11. Selective Transport of Proteins to and from the Nucleus
Nuclear transport receptors are proteins that recognize nuclear localization signals and mediate transport across the nuclear envelope.
Karyopherin is a nuclear transport receptor.
Importins transport macromolecules to the nucleus from the cytoplasm.
Exportins transport macromolecules from the nucleus to the cytoplasm.
How do the nuclear localization sequences serve to send a protein into the nucleus? They are recognized by proteins that function as nuclear transport receptors. Most of these receptors belong to the Karyopherin protein family.
-Members of the Karyopherin family can function as importins that transport substances into the nucleus or exportins that transport molecules out of the nucleus.
-After binding to these Karyopherin receptors, the are then transported through the nuclear pore complex.

12. 9.10 Distribution of Ran/GTP across the nuclear envelope
Ran is one of several types of small GTP-binding proteins that regulate movement of macromolecules through the nuclear pore.
cell4e-fig jpg Movement of these substances through the nuclear pore complex is mediate by a protein called Ran. It mediates both nuclear import and export. Ran is one of several types of small GTP- binding proteins whose conformation and activity are regulated by GTP binding and hydrolysis.
-There are actually lots of cellular proteins that are regulated in this manner such as Ras, the elongation factors that we discussed in chapter 8, Arf, Rac, Rho, etc.
-How does Ran work? The enzymes that stimulate GTP hydrolysis to GDP are localized to the cytoplasmic side of the nuclear envelope, while the enzymes that trigger the exhange of GDP for GTP are localized to the nuclear side of the nuclear envelope. As a result there is an unequal distribution of Ran/GTP across the nuclear pore. The location of the higher concentration of Ran/GTP determines the directionality of the nuclear transport.
-So how does Ran manage to regulate what passes through the nuclear pore complex? Ran regulates the movement through the nuclear pore by controlling the activity of the nuclear transport receptors. Transport though the pore complex begins when a specific importin nuclear transport receptor binds to the nuclear localization signal of the cargo protien in the cytoplasm. The cargo protein-importin complex binds to specific nuclear pore proteins in the cytoplasmic filaments. By sequential binding to more interior nuclear pore proteins, the complex is translocated through the nuclear pore.
-Once inside the nucleus, the cargoprotein/importin complex is disrupted by the binding of Ran/GTP to the importin. This causes a change in conformation to the importin and displaces the cargo protein from the complex and releases it in the nucleus. Then, the importin- Ran/GTP complex is transported back through the pore to the cytoplasm where the GTPase activating protein hydrolyzes the GTP on the Ran to GDP and releases the importin into the cytoplasm where it is free to bind new cargo proteins and participate in a new round of transport.

13. 9.12 Nuclear export
cell4e-fig jpg -In a similar manner, proteins are targeted for export from the nucleus by specific amino acid sequences called nuclear export signals. These sequences are recognized by receptors within the nucleus called exportins that direct protien transport through the nuclear pore complex to the cytoplasm.
-They bind to Ran/GTP promotes the formation of stable complexes between exportins and their cargo proteins. This is the opposite of the import process. Exportins form stable complexes with their cargo protiens in association with Ran/GTP within the nucleus.
-Following transport to the cytosolic side of nuclear envelope, GTP hydrolysis and release of Ran/GDP leads to the dissociation of the cargo protien which is release into the cytoplasm.

14. Regulation of Nuclear Protein Import
The transport of proteins to the nucleus is yet another level at which the activities of nuclear proteins can be controlled.
In one mechanism of regulation, transcription factors associate with cytoplasmic proteins that mask their nuclear localization signals.
The activity of nuclear protiens can be regulated by their transport. For example transcription factors are functional only when they are present in the nucleus. There by controlling their import to and export from the nucleus is one way of regulating gene expression.
-We will talk more about this type of regulation in Chapter 15 when we discuss cell signaling, but for now it is important to understand that regulated nuclear import of both transcription factors and protein kinases has an important role in controlling cell response to an external stimuli. It provides a mechanism for signals received at the cell surface to be transmitted to the nucleus.
-One mechanism of regulation is for transcription factors to associate with cytoplasmic proteins to mask their nuclear localization signals….causing these proteins to remain in the cytoplasm. A good example of this is the trancription factor NfKB.
-NF-kb is maintained as an inactive complex with Ikb which serves to hide or mask its nuclear localization signal, keeping Nfkb in the cytoplasm. However, in response to certain extracellular signals, Ikb is phosphorylated and degraded by ubiquitin-mediated proteolysis. This exposes the NLS on Nf-kb, allowing it to be imported into the nucleus by combining with the proteins Importin. This allows for the Nf-kb to interact with target genes and activate gene expression of those targets.
-A similar process happens with the yeast protein Pho4, except it is maintained in the cytoplasm when the protein is phosphorylated on a residue within the NLS. The phosphate group function to mask the NLS and keeps the Pho4 in the cytoplasm. Signal mediated dephosphorylation allows the protein to be imported into the nucleus.

15. Most RNAs are exported from the nucleus to the cytoplasm.
Transport of RNAs
Most RNAs are exported from the nucleus to the cytoplasm.
RNAs are transported across the nuclear envelope as ribonucleoprotein complexes, or RNPs.
RNA are transported in the oppposite direction of most proteins. Proteins are generally transported from the cytoplasm to the nucleus , while most RNAs are generated in the nucleus are must be exported to the cytoplasm to participate in translation.
-Once again this is an important step in the regulation of gene expression, without tRNA, rRNAs or mRNA translation or protein synthesis is prevented.
-Much like protein import, export of RNAs to the cytoplasm is an energy dependent process requiring nuclear transport recepotors (Karyopherins…exportins) to interact with the nuclear pore complex. They transport most tRNAs, rRNAs and snRNAs in a Ran/GTP dependent manner.
-RNAs are transported across the nuclear envelope as ribonucleoprotein complexes (RNPs). Their export from the nucleus is mediated by nuclear export signals present on proteins within the subunit complex. Pre-mRNAs and mRNAs are associated with a set of at least 20 proteins throughout their processing in the nucleus and transport to the cytoplasm
-There are small RNAs, such as snRNAs and snoRNAs, function within the nucleus as components of the RNA processing machinery. These molecules are recycled during transport so that they can be reused during successive transport events.

16. The Plasma Membrane
All cells—both prokaryotic and eukaryotic—are surrounded by a plasma membrane. This serves to separate the contents of the cell from its environment.
-In this chapter we will focus on the two main roles of the plasma membrane: to protect the contents of the cytoplasm and to mediate interactions between the cell and its environment (mediate cell signals).

17. 13.1 Bilayer structure of the plasma membrane
Studies of the red blood cell plasma membrane provided the first evidence that biological membranes consist of lipid bilayers.
The fundamental structure of the plasma membrane is the phospholipid bilayer, which forms a stable barrier between two aqueous compartments.
Proteins embedded within the phospholipid bilayer carry out the specific functions of the plasma membrane.
cell4e-fig jpg -In most cells today, the plasma membranes are composed of both lipids and proteins. This elecron micrograph of the plasma membrane illustrates that it is a phospholipid bilayer. The two dense lines are the polar phophate heads and the intervening space are the 2 sets of hydrophobic fatty acid tails.
-This membrane serves as a selective barrier to most water soluble molecules. Proteins present in the plasma membrane serve to mediate transport across the memrane.
-What does this mean? It means that the passage of most molecules across the plasma membrane is mediated by proteins. There are proteins present within the membrane that serve as gate keepers to mediate what goes in and comes out of the cell.
-In addition to mediating passage of molecules, proteins in the plasma membrane also have functional roles in cell to cell recognition.

18. The Phospholipid Bilayer
Phosphatidylcholine -- glycerol phospholipid with a head group formed from choline.
Phosphatidylethanolamine -- glycerol phospholipid with a head group formed from ethanolamine.
Phosphatidylserine -- glycerol phospholipid with a head group formed from serine.
Sphingomyelin -- phospholipid consisting of two hydrocarbon chains bound to a polar head group containing serine.
The plasma membrane has been very thoroughly studied and is a fairly well characterized membrane. The plasma membrane of mammalian red blood cells is one model system that has been particularly useful b/c they do not contain nuclei or internal membranes which allows their plasma membrane to be easily isolated for analysis.
-Work done by Gorter and Grendel in the 1920s showed that plasma membranes are lipid bilayers. When they extracted lipids from a known number of RBC with a known surface area, then discovered that the lipids they extracted produced twice the surface area which led them to the conclusion that the membrane was a lipid bilayer and not a monolayer.
-There are 4 major types of phopholipids present in the plasma membrane of animal cells. They are:
-phosphatidylcholine
-phosphatidylethanolamine
-phosphatidylserine
-sphingomyelin
-Collectively these phospholipids account for more than half of the lipids in most membranes. They are distributed between the 2 halves of the membane bilayer with the outer portion of the bilayer containing mostly phophatidylcholine and sphygomyelin and the inner portions consisting of primarily phosphatidylethanolamine and phosphatidylserine.

19. The Phospholipid Bilayer
The fluid mosaic model of membrane structure is now generally accepted as the basic paradigm for the organization of all biological membranes.
Phosphatidylinositol is a phospholipid localized to the inner half of the plasma membrane, which plays an important role in cell signaling.
Glycolipids are lipids consisting of two hydrocarbon chains linked to a polar head group containing carbohydrates.
Cholesterol, a lipid consisting of four hydrocarbon rings, is a major membrane constituent of animal cells.
-Before we talk about the other lipids in the membrane, it is important for you to understand 2 main features of phospholipid bilayer that are critical to its function.
The structure of phospholipids is responsible for the basic function of membrnaes as barriers btw. 2 aqueous compartments.
Bilayers of naturally occuring phospholipids are viscous fluids, not solids. The FAs of most naturally occuring phopholipids have one or more double bonds which serve to introduce kinks into the hydrocarbon chains an make them difficult to pack together. This allows the long hydrocarbon chains to move freely in the interior of the membrane, keeping the membrane soft and flexible. Lipid rafts are clusters of sphingolipids and cholesterol that move laterally within the plasma membrane and associate with specific membrane proteins
-This is why the membane structure is often referred to as the fluid mosaic model.
-What else is present in the lipid bilayer. PI, Glycolipids and cholesterol molecules.
-There is a fifth lipid called phosphatidylinositol present in the inner half of the lipid bilayer. It is present in relatively low concentrations compared to the other phospholipids, but does have an important role in cell signaling which we will discuss later.
-Glycolipids are found exclusively in the outer portion of the plasma membrane with their carbohydrate portion exposed on the surface of the cell. They are also a relatively minor portion of the membrane lipids.
-Cholesterol is a relatively abundant component in the lipid bilayer. It is present in the same molar amount as the 4 other phospholipids.
-Because of its rigid ring arrangement, cholesterol plays a distinct role in membrane structure. Cholesterol molecules insert into the bilayer of phospholipids with it polar hydroxyl group close to the phospholipid head groups. At high temp, cholesterol interferes with the movement of the FA chains, making the outer part of the membrane less fluid. However at low temperatures, it has the opposite effect. By interacting with FA chains it prevents membranes from freezing and maintains membrane fluidity.
-How are cholesterol molecules integrated into the lipid bilayer? Instead of diffusing freely in the PM, cholesterol and sphyngolipids form discrete membrane domains that are referred to as lipid rafts. These serve to cluster sphingolipids and cholesterol to move laterally within the plasma membrane and associate with certain membrane proteins. This permits differerent activities at different temperatures for example.

20. Membrane Proteins
Peripheral membrane proteins are proteins that dissociate from the membrane following treatments with polar reagents that do not disrupt the phospholipid bilayer.
Integral membrane proteins can be released only by treatments that disrupt the phospholipid bilayer.
Transmembrane proteins span the lipid bilayer with portions exposed on both sides of the membrane.
Lipids are the primary structural element of the plasma membrane, while proteins are the major functional unit of the membrane.
-Most plasma membrane consist of 50% lipids and 50% proteins by weight, with carbohydrate portions of glycoproteins and glycolipids making up about 5-10% of the membrane mass. Keep in mind that this is not a one to one ratio because the mass of a protein is much greater the that of a lipid.
-The fluid mosaic model of membrane structure which we discussed in the last slide was introduced in the 1970s and is now generally accepted as the basic organization for all biological membranes. Generally, membranes are viewed as 2-D fluids with protein are inserted into lipid bilayers.
-There are 2 primary classes of membrane associated proteins called peripheral proteins and integral membrane proteins.
-Peripheral proteins are those that dissoicate from the membane following treatments with polar solutions such as solutions with high salt,or Ph that do not disrupt the structure of the lipid bilayer. Once released from the membrane these proteins are soluble in aqueous solutions such as that of the cytosol on the extracellular environment.
-Integral membrane proteins are a part of the membrane. So they can only be released by substances to disrupt or dissolve the phospholipids structure of the membrane. For example, the hydrophobic portion of many detergents displace the membrane lipids and bind to the hydrophobic portions of the integral membrane proteins. This makes the detergent-protein complex soluble in aqeous solutions.
-There is a 3rd type of protein in many membranes that is a subtype of intregral proteins called transmembrane proteins. These proteins span the lipid bilayer with portions exposed to both sided of the membrane. The membrane spanning portions of these proteins are usually alpha helices of 2-=25 hyrdophobic amino acids that are inserted in to the membanr of the ER during synthesis of the polypeptide chain.

21. 13.4 Solubilization of integral membrane proteins by detergents
cell4e-fig jpg This slide shows you an integral membrane proteins and how the membrane must be solubilized with a detegent inorder to release the protein.

22. 13.6 Integral membrane proteins of red blood cells
The most abundant peripheral membrane protein of red blood cells is spectrin, which is the major cytoskeletal protein of erythrocytes.
There are two major integral membrane proteins in red blood cells— glycophorin and band 3.
cell4e-fig jpg Again the membanes of RBCs were used to first understand much of what is known about membranes including the involvement of proteins in membrane structure and function. The membranes of human RBCs contain numerous proteins that were identified by gel electrophoresis of membrane preparations.
-Spectrin is a Peripheral proteins in RBCs. Spectrin is a major cytoskelatal protein in RBCs. Other peripheral proteins include actin, and akyrin.
-Glycophorin and band3 are 2 primary integral membrane proteins in RBCs. As you can see here, band 3 is also an example of a transmembrane protein.
-Glycophorin is a small glycoprotein of 131 amino acids with a molecular wgt of about 30 kDa (half of which is carbohydrate and half is protein). It contains a single 23 amino acid membrane spanning alpha helix and it glycosylated on the amino terminal portion that is exposed on the cell surface.
-Band 3 is an anion transporter proteins that allows the passage of HCO3- (bicarbonate) and Cl- (chloride) ions across the membrane. It is 939 aminot acids and has 14 membrane spanning alpha helices.
-Within the membrane, dimers of band 3 form globular structures that form internal channels through which the ions can travel across the plasma membrane.

23. Membrane Proteins
Some proteins are anchored in the plasma membrane by covalently attached lipids or glycolipids.
Glycosylphosphatidylinositol, or GPI, are glycolipids containing phosphatidylinositol that anchor proteins to the external face of the plasma membrane.
Other proteins are anchored to the inner leaflet of the plasma membrane by covalently attached lipids.
Not all proteins are achored to the membrane by hydrophobic regions present within their sequence.
-A variety of proteins are covalently attached to lipids or glycolipids which serve to anchor the protein to the lipid bilayer.
-Members of one type of these proteins are inserted into the outer leaflet of the plasma membrane by GPI anchors which we have discussed before. GPI anchors are added to certain protiens that have been transferred into the ER and are anchored in the membrane by a C-terminal transmembrane region. The transmembrane region is cleaved as the GPI anchor is added so that the proteins remain attached to the PM only by the glycolipid
-Other proteins are anchored on the inner side of the PM by covalently attached lipids. These proteins are synthesized on free cytosolic ribosomes and then modified by the addition of lipids.

24. 13.10 Mobility of membrane proteins
cell4e-fig jpg -Membrane proteins and phospholipids are unable to move back and forth between the inner and outer leaflets of the membrane at an appreciable rate.
-However, since they are inserted into a fluid bilayer, both proteins and lipids can move or diffuse laterally thorugh the membrane.
-There was some work done in the 1970s by Frye and Edidin, that provided support for the lateral movement of proteins in the fluid lipid bilayer.
-They fused mouse and human cells in culture to generate mouse-human cell hybrids. Then, they analyzed the distribution of proteins in the membranes of these hybrid cells using antibodies that specifically recognize protiens of human and mouse origin. The antibodies were labeled with 2 different flourescent dyes so the human and mouse proteins could be distinguished under the light microscope.
-Immediately after fusion, the mouse and human proteins to segregated to their respective halves of the fused or hybrid cell. However after a 40 minute incubation, the human and mouse proteins had dispersed throughout the cell surface, presumably by lateral diffusion.

25. Mobility of Membrane Proteins
Lipid composition can perturb the free diffusion of membrane proteins.
The basolateral domain is the surface region of a polarized epithelial cell that is in contact with adjacent cells or the extracellular matrix.
The apical domain is the exposed free surface of a polarized epithelial cell.
-Keep in mind that not all proteins are able to freely diffuse. Sometimes their function prevets this from occuring. For example, proteins that are assoicated with the cytoskelaton or proteins associated with other protiens are sometimes restricted from lateral movement
-Red blood cells have a relatively simple membrane structure. Other cells such as epithelial cells are polarized when they are organized into tissues with differient parts of the cell carrying out specific functions.
-The plasma membrane of epithelial cells is divided into the apical and basolateral domains that differ in protein composition and function. For example the epithelial cells in the small intestine absorb nutrients from the digestive tract. The aplical surface faces the intestinal lumen and is covered by microvilli which increase its surface area and facillitate nutrient absorption. The basolateral surface faces the underlying connective tissue and blood supply so it serves it is specialized to transfer the absorbed nutrients to the blood stream.
-To maintain these distinct functions, the mobility of plasma membrane proteins must be restricted to the appropriate domains of the cell surface.
-This partly occurs by the formation of tight junctions which we will discuss in more detail in Ch. 14, but basically the seal the space btw. Cells to block the movement of membrane lipids and proteins. This forces proteins to only diffuse laterally within their apical or basolateral domain, not both.

26. 13.12 Structure of lipid rafts
cell4e-fig jpg -The other way that free diffusion of proteins and lipids is mediated is by the composition of lipids.
-The melting temp. of sphingolipids and phospholipids differ. Sphingolipids tend to cluster with glycolipids into small semisolid patches called lipid rafts. These structures are enriched in cholesteral, GPI anchored proteins and GTP binding proteins.
-The clustering of these molecules prevents lateral movement of some proteins and allows for processes such as endocytosis and receptor mediated signaling to occur.

27. The glycocalyx
The glycocalyx is formed by oligosaccharides of glycolipids and transmembrane glycoproteins.
Selectins are cell adhesion molecules that recognize oligosaccharides exposed on the cell surface.
cell4e-fig jpg -The extracelluar portions plasma membrane proteins are typically glycosylated. Similarly the carbohydrate portion of glycolipids is normally exposed on the outer surface of the plasma membrane.
-The surface of some cells become covered with a carbohydrate coat called to glycocalyx. It is formed by the oligosaccharides of glycolipids and transmembrane glycoproteins.
-The glycocalyx serves to help protect the cell surface and helps maintain contact with other cells.
-Along with the glycocalyx, selectins help with cellular interactions. E-selectin is a transmembrane protein expressed by endothelial cells that binds to an oligosaccharide expressed on the cell surface of leukocytes. The oligosaccharide recognized by E-selectin contains N-acetylglucosamine, fucose, galactose and sialic acid.

28. 13.15 Permeability of phospholipid bilayers
The internal composition of the cell is maintained because the plasma membrane is selectively permeable to small molecules.
Only small, relatively hydrophobic molecules are able to diffuse across a phospholipid bilayer at significant rates by using passive diffusion.
Passive diffusion is the simplest mechanism by which molecules can cross the plasma membrane.
Specific transport proteins mediate the selective passage of small molecules across the membrane, allowing the cell to control the composition of its cytoplasm.
cell4e-fig jpg The next several slides focuses on transport of molecules across the lipid bilayer of most cells.
-We mentioned earlier that movement of molecules into and out of cells is mediated by the selective permeability of the plasma membrane. Most molecules are not able to diffuse through the phospholipid bilayer causing the PM to serve as a barrier between the cytosol and the extracellular environment of the cell.
-Only very small hydrophobic molecules such as O2 and and water….this is referred to as passive diffusion. The molecules seem to dissolve in the lipid bilayer in order to get from one side of the membrane to the other.
-Larger molecules like glucose, amino acids and charged ions all required proteins and energy to be transported across the phospholipid bilayer. These molecule cannot make their way across the hydrophobic interior of the membrane.
-Transport and Channel proteins seem to control the traffic of most molecules into and out of the cell.

29. Phagocytosis
Phagolysosomes, which are phagosomes fused to lysosomes, contain lysosomal acid hydrolases that digest the ingested material.
The ingestion of large particles by phagocytosis plays distinct roles in different kinds of cells.
-The ingestion of large particles by phagocytosis plays distinct roles in different kinds of cells. Amoebas use phagocytosis to capture food particles which is what you see in part A, while macrophages (immune cells in humans) use phagocytosis to engulf and destroy pathogens.

30. Receptor-Mediated Endocytosis
Receptor-mediated endocytosis, a form of pinocytosis, provides a mechanism for the selective uptake of specific macromolecules.
Clathrin-coated pits are specialized regions of the plasma membrane where specific cell surface receptors are found.
Dynamin, a membrane-associated GTP-binding protein, assists in the budding off of pits from the plasma membrane.
In addition, there is a specific type of endocytosis called receptor mediated endocytosis which allows for the selective uptake of specific macromolecules.
-First the macromolecule or ligand (Ligands are molecules that bind to a receptor) must bind to specific receptors on the cell’s surface. Most of these receptors are concentrated in specialized regions of the plasma membrane called clatrhin coated pits.
-A membrane associated GTP binding proteins called Dynamin helps the coated pits to bud out of the membrane to form small clathrin coated vesicles.
-Then the clathrin coated vesicles fuse with early endosomes in which their contents are sorted for transport to lysosomes or recycling to the plasma membrane.
-The uptake of cholesterol by mammalian cells has provided a great model for understanding receptor mediated endocytosis. Cholesterol is transported in the bloodstream in the form of lipoproteins….LDL and HDL. Research has shown that the uptake of LDL by mammalian cells to use in various biological processes required the binding of LDL to specific cell surface receptors concentrated in clathrin coated pits. The LDL molecules are then taken into the cell by endocytosis. Individuals with the disease familian hypercholesteremia have a mutated version of the LDL receptor. As a result cholesterol is not taken up by their cells very efficiently, so it accumulates in their blood stream causing high cholesterol and the ensuing diseases.

31. Receptor-Mediated Endocytosis
Clathrin assembles into a basketlike structure that distorts the membrane, forming invaginated pits.
Clathrin coated pits occupy about 1-2% of the surface area of plasma membranes.
In eukaryotic cells there are more than 20 different receptors to selective internalize molecules using this type of pathway.
-Adaptor proteins bind to clathrin and to the internalization signals present in the cytoplasmic tails of the receptors, creating this basket like structure or clathrin network that exists on the surface of the membrane.
-Most clathrin coated pits have a lifetime of about 1-2 minutes which results in internalization of an area equivalent to the entire PM every 2 hours.

32. Receptor-Mediated Endocytosis
Caveolae are small invaginations of the plasma membrane.
Caveolin is a protein that interacts with lipid rafts and forms caveolae.
Macropinocytosis is a process where large vesicles can mediate the uptake of fluids.
In addition to clathrin mediated pathways of endocytosis, there are also clathrin independent pathways.
-One such pathway involved the uptake of molecules in caveolae ro small invaginations of the PM that are organized by caveolin.
-Caveolins are a family of proteins that interact with cholesterol molecules in lipid rafts, insert into the cell membrane and interact with one another to form the structure of the caveolae.
-Caveolae carry outs receptor mediated endocytosis by way of specific transmembrane receptors.
-Macropinocytosis is another non-clathrin mediated type of endocytosis.

33. Protein Trafficking in Endocytosis
Endosomes are vesicles with tubular extensions, located at the periphery of the cell, that fuse with clathrin-coated vesicles which have shed their coats.
An important feature of early endosomes is that they maintain an acidic internal pH as the result of the action of a membrane H+ pump.
We talked about this in Chapter 10, so we are not going to review it again. You should take some time to ensure that you are familiar with the process.

34. Protein Trafficking in Endocytosis
Recycling to the plasma membrane is the major fate of membrane proteins taken up by receptor- mediated endocytosis.
Ligands and membrane proteins destined for degradation in lysosomes are transported from early endosomes to late endosomes, which are located near the nucleus.
Receptor down-regulation is a phenomenon where receptor-ligand complexes are removed from the plasma membrane, thereby terminating the response of the cell to growth factor stimulation.
Something else to consider about all of these receptor or membrane protein mediated processes used to bring substances into or out of a cell……what happens to the receptor or membrane protein that mediates the process?
-Recycling to the PM is the ultimate destination of membrane proteins taken up by receptor mediated endocytosis. This occurs after the receptor dissociates from it bound ligand within the early endosomes.
-This allows for continuous internalization of their ligands. For example each LDL receptor makes a round trip from the PM to the endosomes and back again about every 10 minutes.
-This figure shows you how synaptic vesicles are recycled which involves clathrin coating after release from the endosome.
-Ligands and membrane protiens destined for degradation in lysosomes are tranported from early endosomes to late endosmes which eventually become lysosomes. Within the lysosomes the endocytosed materials are degraded by the action of acid hydrolases.
-Not all receptors are recycled to the plasma membrane….some are tranported to lysosomes and degraded along with ligands. For example the cell surface receptors for certain growth factors are internalized following growth factor binding and eventually degraded in lysosomes. What is the end result of this occuring? It removes the receptor-ligan complexes from the PM which prevents the cell from responding to further GF stimulation. This is called receptor down regulation.