Cell Structure Lecture 2 Sep 4

Question 1

How are lipids and proteins meant for the plasma membrane and organelles other than the ER delivered?

Answer 1

Through membrane flow.

Membrane flow involves the ER, the Golgi, the lysosomes, and the plasma membrane

Question 2

What is the overall direction of membrane flow?

Answer 2

Membrane from the ER flows to the golgi.

Membrane from the golgi can flow to multiple places:

1. back to the ER
2. Out to the plasma membrane in vesicles
3. To the endosomes to form lysosomes
4. To other areas of the cell in secretory vesicles

Question 3

What overal mechanism does membrane flow happen through?

Answer 3

vescicles budding off and fusing with new membranes.

This requires fusion of lipid bilayers, and thus maintains the topological orientation of membrane proteins and lipids.

Question 4

Familiar hypercholesterolemia (FH) is an example of a specific deficit in an endocytic pathway.

Describe it.

Answer 4

Patients have a defect in the cell’s ability to endocytose cholesterol as LDL particles, so the cholesterol builds up in the blood and you get the deposition of atherosclerotic placques.

Question 5

Which face of the golgi receives vesicles? which face of the golgi release vesicles?

Answer 5

The cis face (closest to the ER) receives vesicles.

The contents of those vesicles are modified as they pass through the golgi. Some of them will stay and be luminal golgi proteins. Others are meant for transport elswhere.

These proteins will leave in vesicles being released from the trans face.

Question 6

What sorts of modifications do proteins undergo as they pass through the golgi?

Answer 6

Any N-linked glycosylation they have will be trimmed or added to.

O-linkage glycosylation will occur in the golgi

Question 7

What are the 7 functions of the golgi?

Answer 7

1. Receives lipid and protein products from the ER
2. Returns “escaped” proteins that should be resident in the ER
3. Modifies glycoproteins (both trimming and addition)
4. Solfation and other post-translational modifications
5. Glycolipid and sphingomyelin production
6. Add O-linked oligosaccharides to proteoglycans
7. Directs materials to be transporte dfurther along 3 primary routes: lysosomes, secretory vesicles, plasma membrane

Question 8

What protein coat is used in transport from the golgi out to the plasma membrane or to the lysosomes?

What protein coat is used in transport from the ER to the golgi?

What protein coat is used in transport rom the Golgi back to the ER and intra-golgi transport?

Answer 8

Golgi to plasma membrane or lysoeoms uses clatherin

ER to golgi uses COP2

Golgi to ER or intra-golgi uses COP1

Question 9

What is the series of steps for COP1 and COP2 vesicle formation?

Answer 9

This utilizes small G proteins: Sar1 for COP2 and Arf1 for COP1

The small G proteins will be turned off when they’re attached to GTP and turned off if they’re attached to GDP.

GEFs catalyze the switch from GDP to GTP, activating the small G proteins. GAPs hydrolyze the GTP to GDP, inactivating the small G proteins.

So in COP2:

1. a GEF catalyzes the GDP to GTP switch, activating Sar1.
2. Sar1 then extends a ydrophobic tail that inserts into the lipid bilayer
3. Sar1 then recruits other proteins including Sec23 and Sec24. Sec 24 binds transmembrane receptors for specific cargo proteins–these are the protein taht will be in the vesicle for transport
4. THe binding of these proteins results in a conformational change that begins to curve the membrane
5. Sec13/31 is then recruited, which forms a cage-like structure, helping the besicle to further form and pinch off.

Question 10

How does the golgi know which proteins to send back to the ER?

Answer 10

Resident ER proteins will all have a specific amino acid sequence called the KDEL.

KDEL receptors in the golgi membranes bind these proteins, which are then packaged into vesicles for return to the ER using COP1.

Question 11

What three proteins play critical roles in the accurate delivery and fusion of vesicles?

Answer 11

Rabs

Rab effectors

SNAREs

Question 12

How does vesicle docking occur with selectivity?

Answer 12

The selectivity comes from the Rabs. Different Rabs bind to different membranes, thus serving as markers of membrane types.

Vesicles with a specific Rab will only dock on membranes that have the same Rab.

SNARES come in pairs as well, with v-SNARES only interacting with their favorite t-SNARES

Question 13

What protein actually mediates the fusion of a vesicle to the membrane?

Answer 13

SNARES

the vSNARE is on the vesicle and the tSNARE is on the target membrane.

Once the vesicle gets close enough, the v and t SNARES will wrap around each other and pull the vesicle in.

Question 14

Besides Rabs and SNARES, what is another way membrane targets are identified for vesicle formation and fusion?

Answer 14

Throguh the lipid concent of the membrane

Specifically, the content of specific phosphotidylinositol species can serve to identify different membranes and membrane domains.

Question 15

Why are proteins modified in different ways at diferent locations in the Golgi as they pass through?

Answer 15

Because enzymes are arranged differently from the cis faces through to the trans face.

Question 16

How does the golgi know to send lysosomal hydrolytic enzymes to the lysosome?

Answer 16

The hydrolytic enzymes are all tagged with a mannose 6-phosphate on the oligosaccharide chain.

There are receptors in the golgi membrane trans face that will bind to the mannose 6-phosphate, concentrate the hydrolytic enzymes together in a vesicle, and use the coating protein clatherin to bud off a secretory vesicle to go to the lysosome.

Question 17

What are lysosomes? WHat is their function?

Answer 17

They are membrane-enclosed compartments that are filled with hydrolytic enzymes; they carry out the intracellular digestion of numerous cellular materials.

They have proteases, nucleases, glycosidases, lipases, phospholipasea, etc.

All are ACID HYDROLASES, meaning they’re only active at low pH.

Question 18

What safety mechanism does the body use to protect itself just in case the hydrolytic enzymes get spilled out form the lysosomes?

Answer 18

The hydrolytic enzymes are active only at low pH, and when they’re being transported in vesicles from the golgi tot he lysosome, the vesicle packages them at neutral pH so they’re not active until they reach the lysosome.

Question 19

What are some forms of specialization that the lysosomal membrane has?

Answer 19

1. the membrane is highly glycosylated for protection
2. it contains proton pumps (H+ ATPases) to create an acidic lumen
3. contains numerous transporters to shuttle breakdown products (sugars amino acids and nucleotides) to be used again in the cytoplasm

Question 20

How do lysosomes lower the pH of their lumen to activate the hydrolytic enzymes?

Answer 20

proton pumps to pump in H+ and make the environment more acidic

Question 21

Before the lysosome receives something to eat and hydrolytic enzymes to eat it with, what is it called?

Answer 21

an endosome

Question 22

What are three paths through which things can enter the lysosome?

Answer 22

1. Autophagy (breaking down old organelles0
2. Phagocytosis of big stuff like bacteria and dead cells by large specialized endosomes called phagosomes: macrophages and neutrophils.
3. Endocytosis. THe VAST majority of vesicles that bud into the plasma membrane carry contents that are brought directly to the lysosome to be broken down.

Question 23

What happens to the receptors for the cargo proteins that were brought into the lysosome to be digested?

Answer 23

They’re repackaged in vesicles and then exocytosed back out to the membrane they came from (ER or plasma, etc.) so they can pick up more of the same cargo

Question 24

After the mannose 6-phosphate receptor drops off it’s hydrolytic enzymes into the endosome, the mannose 6-phosphate is repackaged in a vesicle to undergo retrograde transport back to the golgi.

What coat protein does this?

Answer 24

Clatherin does the golgi to lysosome, but the reverse is through retromer coat proteins. Think retrograde = retromer.

Question 25

What are the two forms of endocytosis?

Answer 25

Pinocytosis (cell drinking)

Happens in most cells, a few different type sof vesicles are involved, receptor mediated endoctosis can concentrate specific moleucles for internalization

transcytosis is a special case of this that involves the movement of material straight thorugh cells from the apical side fo the basal lateral side–this skips the lysosome

Phagocytosis is carried oout by only macrophages and neutrophils–it’s ingestion of large particles or bacteria.

Question 26

Edocytotic vesicles can either be coated or uncoated.

What coat protein do the coated endocytotic vesicles have?

What protein is associated to uncoated endocytotic vesicles as a permament integral membrane protein?

Answer 26

Coated endocytotic vesicles contain clathrin as a transient perpheral membrane protein.

Uncoated vesicles often contain caveolin as a permanent integral membrane protein

Question 27

Uncoated endocytotic vesicles, or caveolae, are enriched where in the membrane?

How do they look on an electromicrograph of a cell that has been slam frozen?

Answer 27

On lipid rafts, and they are therefore important for signalling events in addition to bringing things into the cell.

They look like honeycomb on the outside of cells.

Question 28

What are the steps for clatherin and receptor-mediated endocytosis?

Answer 28

1. Adaptor proteins bind receptor-ligand complexes
2. Clathrin assembles onto the adaptor proteins, deforming the membrane and forming a coated pit.
3. The vesicle invaginates and the dynamin pinches off the neck of the vescicle
4. Uncoating ATPases remove the clathrin coat, which is then recycle
5. The endocytosed vesicle travels to its target membrane.

Question 29

Of the coating proteins we’ve talked about, which is the thickest?

Answer 29

clatherin

Clatherin self associates to form a trimer of heavy and light chains called a triskelion.

Triskelions spontaneoulsy assemble to form a bascket-like coat whihc deforms the membrane and facilitates vesicle invagination.

Question 30

Why must all cells carry out constitutive secretion?

What is the “signal” for something to be constitutively secreted to the plasma membrane?

Answer 30

They need to renew their plasma membranes and balance the quantity of membrane lost in exocytosis and gained in endocytosis.

It appears to be the default pathway. In other words, the materials in the Golgi WITHOUT any signals routing them to the lysosomes or secretory granules will automatically be exocytosed through the plasma membrane constitutively.

Question 31

Give an example of a cell undergoing the regulated pathway of exocytosis of material?

Answer 31

The cell will hang onto the material in a secretory vesicle until it receives the signal to release it.

Mast cells hold their histamines in secretory granules near the surface of the plasma membrane.

A spike in cytoplasmic Ca++ is usually the signal for the secretory granules to fuse with the plasma membrane, open and release the histamine into the extracellular space.

Question 32

WHere do mitochondria tend to localize in the cell?

Answer 32

Mitochondria form a dynamic network

Many are located along microtubules (because they’re motile and need to travel around in the cell) and often establish close connections with the ER (because they need to regenerate their membranes)

They will also be in high concentration in areas that need more ATP for energy, such as ATP dependent ion pumps (salivary glands), cilia (like sperm tails) and cardiac muscle

Question 33

How do mitochondrial proteins encoded in nuclear DNA get into the mitochondria?

Answer 33

They DO NOT use vesicular transport!!!

A signal sequence on the polypeptide (not a specific sequencethough–an alpha helix structure with hydrophobic residues along one side of the helix) is recognized by transporters in the mitochondrial membranes (TOMs and TIMs)

CHaperones help the polypeptides bind to the TOM on the outer membrane and spool into the intermembrane space

As it is spooling, if it is meant to be in the matrix, the polypeptide will bind to a TIM on the inner membrane and spool inside.

Question 34

TIMs and TOMs get mitochondrial proteins intot he mitochondria from the cytoplasm.

What translocator works in the opposite direction to transport proteins synthesized in the matrix outward?

Answer 34

OXA translocators

Question 35

How do mitochondria get/replenish their membrane lipids?

Answer 35

Many lipids are removed from the ER and inserted into mitochondrial membranes by exchange proteins.

This is why mitochondria are often located near the ER

Question 36

WHat are peroxisomes?

What is their function?

How are they produced?

Answer 36

They are spherical structures bound by a single membrane.

THey are involved in many oxidation reactions like making and destroying hydrogen peroxide.

They also take part in detoxification reactions (oxidation of ethanol to acetaldehyde), beta-oxidation of fatty acids, and formation of myelin phospholipids

They’re produces through hybrid budding from the ER, import of peroxisomal proteins made by free ribosomes, and the addition of a three-AA import signal in the C terminal end.

Question 37

WHat comprises the nuclear envelope?

Answer 37

1. outer nuclear membrane
2. inner nuclear membrane

and underneath all that is the:

nuclear lamina

Question 38

What comprises the nuclear matrix?

What is the purpose of the nuclear matrix?

Answer 38

The nuclear matrix is comprised of filamentous scaffolding made up of lamins and RNA

It provides site of attachment for specific spots along chromosomes, which can play a role in gene expression regulation

Question 39

What areas of the chromosome are specialized to bind to the nuclear matrix?

How does this affect how chromatin is organized in the nucleus?

What replication enzyme is in high concentration near the MARS/SARS?

Answer 39

MARS or SARS

This means the chromatin is held in loops danging from the MARS attachments on the chrosome scaffolding. These are the loop domains, which range from about 50,000 to 200,000 nucleotide pairs long (note that some genes are longer than 200,000 NT pairs, so they are held on multiple loops).

Toposiomerase activity is increased because there is higher levels of torsional stress where the connection is.

Question 40

What is known about where chromosomes reside in the nucleus?

Answer 40

Individual chromosomes tend to occupy discrete territoried in the nucleus.

Question 41

What is comprises the most obvious nuclear structure?

Answer 41

The nucleolus!

This is a large aggregat ot macromolecules including rRNA genes, precursor rRNAs, Matrue rRNA, rRNA processing enzymes, ribosomal proteins, and partially assembled ribosomes

It’s dark because a very large number of rRNA molecules need to be produced to help construct all the ribosomes a cell needs.

Question 42

How has the cell adapted to the necessity of making many rRNA molecules to construct enough ribosomes for the cell?

How is this related to the nucleolar orgnizing regions?

Answer 42

THe cell needs a lot of ribosomes, so the rRNA genes have duplicated and many copies exist in clusters along 10 chromosomes.

The clusters are called nucleolar orgnizing regions

they come together, attract the related proteins and RNA machinery for ribosome production and form the nucleolus

Question 43

What is the purpose of the nuclear lamina?

What proteins make up the lamina?

Answer 43

The nuclear lamina lies just underneath the inner membrane. It is a proteinaceous meshwork of filaments composed of a protein called lamin, which is a member of the intermediate filament family of proteins.

Question 44

How does the nucleus get things like mRNA out into the cytoplasm?

Answer 44

Through nuclear pores.

Typical cells contain a few thousand nuclear pores which are assemblies of nucleoporin proteins.

Question 45

What particles can pass through nuclear pores without active transport?

Answer 45

Anything smaller than 5000 daltons or less can pass through with ease

Things between 5,000 and 60,000 daltons can still make it through, but the larger it is the slower it will go.

Anything over 60,000 daltons will require facilitated transport.

Question 46

Where are nuclear proteins made? How do they get into the nucleus?

Answer 46

Like all proteins, nuclear proteins are made in the cytoplasm.

Nuclear proteins will have a nuclear localization signal (NLS) which is a short region of amino acids that are lysine rich.

The NLSs are recognized by nuclear transport receptors which bind to both the cargo and to the nuclear pore. These will then drag the cargo into the pore.

Export is similar

Question 47

How is the directionality of transport into or out of the nucleus directed?

Answer 47

Through a small G protein called Ran.

For imported proteins: Ran binds import receptors in the presence of GDP and dissociates in the presence of GTP

FOr exported proteins, Ran binds export receptors in the presence of GTP and dissociates in the presence of GDP.

Concentration of GTP is high in the nucleus, so the Ran bound to import receptos will release them in the nucleus

Concentration of GDP is high in the cytoplasm, so the Ran bound to export receptors will release them in the cytosol.

Question 48

Why is the concentration of GDP high in the cytosol and the concentration of GTP high in the nucleus?

Answer 48

Because a Ran-GEF (exchange factor) is concentrated in the nucleus, while Ran-GAP (activating protein) is concentrated in the cytoplasm.

This makes the cytoplasm high in Ran-GDP but low in Ran-GTP

The nucleus is high in Ran-GTP and low in Ran-GDP