Cell Biology Test 3

Front 1

Describe the 6 different pathways that sort proteins.

Back 1

1. If the protein contains no targeting sequence, then it will be sent to the cytosol
2. It can be sent to the ER
3. It can be sent to the mitochondria
4. It can be sent to the chloroplast
5. It can be sent to the nucleus
6. It can be sent to the peroxisome.

Front 2

Describe the 6 kinds of targeting sequences.

Back 2

1. ER --- N-terminal
2. Mitochondria --- N-terminal
3. Chloroplast --- N-terminal
4. Peroxisome --- C-terminal
5. Nucleus --- Internally located
6. Internal “Stop Transfer”

Front 3

Where are secretory proteins made?

Back 3

They are made on ER-bound ribosomes (rough ER).

Front 4

What are the two key structures that act together with the N-terminal signal sequence to target a protein to the ER membrane?

Back 4

The signal-recognition particle (SRP) which transiently binds simultaneously to the ER signal sequence, and its receptor on the ER membrane.

Front 5

Describe the SRP and its receptor.

Back 5

The SRP is a ribonucleoprotein complex; the receptor is an integral membrane protein made up of two subunits, α and β. It acts to not only help mediate interaction of a nascent secretory protein with the ER membrane, but also acts to transfer the ribosome with its nascent polypeptide chain to the transmembrane translocon (Sec 61).

Front 6

What is cotranslational translocation?

Back 6

The translocation of a secretory protein into the ER lumen that occurs while the translocon-bound ribosome translates the mRNA encoding the nascent protein.

Front 7

What is a translocon?

Back 7

A protein-lined channel within the ER membrane. The elongating chain passes directly from the large ribosomal subunit into the central pore of the translocon (donut with a gate).

Front 8

What is needed to open the gate of the translocon and allow the protein to enter?

Back 8

The hydrolysis of GTP

Front 9

What happens when the ribosome has finished translating the mRNA for the secreted protein?

Back 9

The ribosome is released, the remainder of the protein is drawn into the ER lumen, the translocon closes, and the protein assumes its native folded conformation with the help of chaperones.

Front 10

How does the ER hold on to the incoming protein?

Back 10

BiP-ATP binds to entering segments of the polypeptide and prevents the backwards movement out of the ER. Once the peptide is in the ER, the ADP molecules that were bound to the peptide fall off-“ratchet effect”

Front 11

Where are integral membrane proteins synthesized and how are they oriented?

Back 11

They are made on the membrane of the rough ER – if the N-term is in the internal lumen of the ER, then when it is sent to the plasma membrane, the N-term will be on the external face.

Front 12

What end of the peptide enters the ER for type I integral membrane proteins?

Back 12

The N-terminus enters first (remember, the N-term will be on the external face of the cell). Such proteins are synthesized with a hydrophobic N-terminal signal sequence like secretory proteins, but then also contain an internal STA sequence (“stop-transfer anchor sequence”).

Front 13

What end of the peptide enters the ER for type II integral membrane proteins?

Back 13

The N-terminus stays in the cytosol, and the rest of the protein loops through the translocon as it is being synthesized, until an internal signal anchor enters the translocon and then partitions into the adjacent lipid bilayer. The ribosome continues translating the mRNA, such that the remaining C-terminal portion of the protein passes into the lumen of the ER. Consequently, C-terminus of the protein ends up in the lumen of the ER (remember, the C-term will then be on the external face of the cell)

Front 14

What is the “stop-transfer anchor sequence” or STA sequence?

Back 14

It is the internal hydrophobic part of the peptide sequence that will be the transmembrane part of the integral membrane – it is a roughly 20 amino acid hydrophobic sequence that forms an α-helix in the membrane.

Front 15

How do type II integral proteins keep the N-terminus from entering the ER?

Back 15

The peptide has positively charged side chains to keep the N-term on the cytosolic side

Front 16

How does methotrexate affect dihydrofolate reductase and what is this used for?

Back 16

In the absence of methotrexate, the DHFR would be able to unfold and pass through the pore structure in the mitochondrial membranes. When methotrexate is present in the cytosol, it binds irreversibly to the active site of DHFR, locking it into a folded conformation. When this happens, it gets stuck in the pore. This was used to locate pores in electron microscopy studies to locate the pores in mitochondrial membranes.

Front 17

Describe adrenoleukodystrophy and Lorenzo’s oil.

Back 17

It is characterized by accumulation of very long chained fatty acids, especially in the Schwann cells that create the myelin sheath around axons. It is caused by a mutation in the ABCD1 gene also known as the ALD gene. This gene codes for a pump that transports very long chain fatty acids into the peroxisome to be degraded. Lorenzo’s oil was created to treat this condition for a boy that had this. Since oleic acid and erucic acid inhibit the enzyme responsible for the synthesis of very long chain fatty acids, this became a treatment of this disease.

Front 18

How is a constitutive secretory protein different from a regulated secretory protein?

Back 18

A constitutive one is released immediately after synthesis.

Front 19

How is the knowledge about how insulin is synthesized different today from what was believed in the 1970’s?

Back 19

It was not known that there was a signal sequence located on the N-term of the proinsulin molecule; the precursor with the signal sequence is called “preproinsulin”. For an insulin molecule to be considered mature, it must have the signal sequence cleaved and have an internal sequence cleaved off after the disulfide bonds form. The internal peptide sequence is removed on the way to the plasma membrane.

Front 20

Describe a signal sequence.

Back 20

Usually 6-12 hydrophobic amino acids long, proceeded by 1 or more positively charged amino acids. This is what the SRP recognizes.

Front 21

How is the speed of translation changed when the SRP binds to the signal sequence?

Back 21

It is greatly slowed or stopped.

Front 22

If an integral protein is being made on the ER membrane with the N-terminus on the luminal side and the C-term on the cytosolic side, which terminus will end up on the external side of the plasma membrane?

Back 22

N-terminus

Front 23

What end of the type 3 transmembrane proteins is located in the ER lumen?

Back 23

N-terminus, just like type 1

Front 24

Describe type 4 integral proteins.

Back 24

They are multi-pass proteins which can have their N- or C-term on either side of the plasma membrane

Front 25

What environments in the cell are oxidizing and which are reducing?

Back 25

Cytosol is reducing, ER lumen is oxidizing

Front 26

Describe glutathione.

Back 26

It is a reducing agent. The oxidized form of it, GSSG, creates disulfide bonds in the ER lumen (acting together with PDI and Ero1).

Front 27

What are the types of membrane proteins?

Back 27

- Types 1-4
- GPI-anchored

Front 28

How are the GPI-anchored synthesized?

Back 28

They start out just like type 1’s with the N-term on the ER luminal side. GPI transamidase then transfers the luminal N-term end onto a GPI preformed anchor attached to the membrane.

Front 29

What happens to proteins in the lumen of the ER?

Back 29

- they are assisted in folded correctly including disulfide bond formation, proline peptide bond isomerization and multi-chain oligomerization
- they get glycosylated

Front 30

What are two functions of BiP?

Back 30

Chaperoning and post-translational translocation across the ER membrane (ratchet)

Front 31

What does calnexin do and how?

Back 31

When an unfolded protein is released in to the ER through a translocon, it gets glycosylated by oligosaccharide glycosyl transferase which transfers a complex precursor oligosaccharide onto certain asparagines residues. This complex precursor oligosaccharide ends in a short chain of 3 glucose residues. As the protein folds, these glucose residues are sequentially removed. These glucose residues are recognized by the integral membrane protein chaperone calnexin as a signal that the protein needs to be folded. The protein gets folded and is released. If the protein was folded correctly, then the glucose residues will be removed and the protein will be released from the ER; if not, a glucosyl transferase enzyme recognizes the protein as still unfolded, and re-glucosylates it; whereupon the Calnexin will have another go at folding it.

Front 32

What enzyme catalyzes the formation of disulfide bonds in the ER?

Back 32

Protein disulfide isomerase (PDI)

Front 33

What enzyme is used to allow rotation of the C-N bond in a proline peptide bond?

Back 33

Peptidyl-prolyl isomerase

Front 34

On what end of an ER-resident protein is the KDEL sequence?

Back 34

The C-terminus

Front 35

Describe the function of the KDEL sequence.

Back 35

This is found on proteins that are supposed to reside in the ER lumen after synthesis. If by chance a protein with the KDEL sequence is packaged into vesicles released from the ER, the golgi will send it back to the ER.

Front 36

What does brefeldin A do?

Back 36

It blocks the formation of a transport vesicle

Front 37

What does nocodazole do?

Back 37

It causes depolarization of microtubles-without these microtubules, transport vesicles cannot travel in the cell surface receptor

Front 38

What are the two kinds of linkages of oligosaccharides to proteins?

Back 38

- O-linked – attaches to an OH on the side chain of serine or hydroxylysine
- N-linked (complex) – attaches to an N on certain asparagines

Front 39

Are sugars added one at a time or all at once?

Back 39

One at a time

Front 40

What molecule is usually found at the terminal sequence of a glycosyl addition and what is unique about this?

Back 40

NANA (sialic acid), it is negatively charged

Front 41

What is the sugar donor when glycosylating proteins?

Back 41

Nucleotide-sugar precursors

Front 42

Where are these Nucleotide-sugar precursors made and how are they used in glycosylation

Back 42

They are made in the cytosol, but glycosylation happens in the ER lumen by glycosyl transferase, so they must be transported into the lumen of the ER by an antiporter that will let in the sugar containing nucleotide (UDP) while letting out the non-sugar nucleotide (UMP)

Front 43

Where in the cell are N-linkages and O-linkages final stage occurring to proteins?

Back 43

- N-linked – in the golgi (slides 47 and 48)
- O-linked – in the golgi (slide 42)

Front 44

What are the 3 types of N-linked oligosaccharides? (slide 48)

Back 44

- high mannose
- hybrid
- complex

Front 45

What is common to high mannose, hybrid, and complex N-linked oligosaccarides ?

Back 45

They share a common core (slide 43)

Front 46

What lipid is the “common core” of N-linked oligosaccharides synthesized from?

Back 46

Dolichol pyrophosphate (slide 44)

Front 47

What is unique about dolichol? (slide 45)

Back 47

It starts to make the glycosylated sugar precursor on the cytosolic side of the ER membrane, and then flips it to the luminal side to complete its synthesis

Front 48

How does the oligosaccharide donate its sugars to a protein? (slide 46)

Back 48

As the protein is being synthesized and fed through the translocon into the ER lumen, the dolichol membrane protein is watching for the correct amino acid sequence. When it encounters an asparagine followed by [x] (any aa) followed by a serine, it knows to attach the precursor sugar to the asparagine.

Front 49

How are mannose-6-phosphate tagged enzymes transported?

Back 49

Via clathrin-coated vesicles

Front 50

How is it that hydrolytic enzymes are transported to the lysosome without causing any damage to surrounding structures?

Back 50

The intracellular vesicles are at pH 7.2. At this pH, they are inactive. They are only active at pH 5 which is only in the lysosome.

Front 51

What does a mannose-6-phosphate tag do?

Back 51

It is a tag that is added in the golgi to an enzyme destined for the lysosome. These enzymes are sent out in a clathrin-coated vesicle to the cytosol where they fuse with a late endosome. This will fuse with the lysosome to release the enzymes. Enzymes such as nucleases, proteases, lipases, phospholipases, etc. are sent there.

Front 52

How does a mannose-6-phosphate tag get attached?

Back 52

The protein gets glycosylated to contain a high mannose N-linked oligosaccharide. This gets phosphorylated by GlcNAc phosphotransferase. Phosphoglycosidase then cleaves the donor nucleotide to leave just the sugar attached to the high mannose. This is the mannose-6-phosphate.

Front 53

What is I-cell disease?

Back 53

It is a defect where mannose-6-phosphate tags are not added to lysosomal enzymes (cells do not contain GlcNAc phosphotransferase. Since thelysosomal degradation enzymes are therefore secreted rather than delivered to the lysosome, debris and toxins are not properly degraded.

Front 54

What happens to these tags once the enzyme reaches the lysosome?

Back 54

They are removed.

Front 55

What is clathrin?

Back 55

A triskelion (3-chain) molecule that is fibrous polymerizes to from a polygonal cage that encases the vesicles that bud off the Golgi and certain endocytic vesicles that form on the plasma membrane. Each leg of the clathrin contains 3 heavy chains and 3 light chains. These polymerize to form a lattice with an intrinsic curvature.

Front 56

What does dynamin do?

Back 56

It is a cytosolic protein that polymerizes around the neck portion of nascent clathrin-coated vesicles and then hydrolyzes GTP. The energy derived from GTP is thought to drive “contraction” of dynamin around the vesicle neck until it pinches off.

Front 57

How is an LDL particle organized?

Back 57

It is a ball of cholesterol that forms a single phospholipid layer. A bilayer is not necessary because the tails can point inwards and have hydrophobic interactions with each other.

Front 58

What is a cholesterol ester and why do we have them in this form?

Back 58

A cholesterol with a long fatty acid attached to hydroxyl group. This is how it is transported.

Front 59

How do certain viruses gain access to our cytosol once engulfed in an endosome?

Back 59

The coat proteins (envelope proteins) of certain viruses mediate binding of the virus to the cell surface (to cellular molecules that are part of clathrin coated pits) (receptor-mediated endocytosis pathway). The virus particles thereby become located in late endosomes (sometimes termed “CURL” compartment), via the usual pathway of receptor-mediated endocytosis. The acidic pH of the endosome causes a conformation change in the virus coat proteins. [This cannot happen at pH 7.2.] The hydrophobic tips of the viral coat proteins insert themselves into the membrane of the endosome. This causes the fusion of the viral membrane and endosome membrane, permitting access of the viral nucleic acid to the cytosol.

Front 60

What 2 other proteins polymerize to form shells that initiate vesicle formation besides clathrin?

Back 60

- coatamer protein 1 (COP-1)
- coatamer protein 2 (COP-2)

Front 61

What is the difference between COP-1 and COP-2?

Back 61

COP-2 carries cargo in an anterograde direction, ie, from the ER to the golgi, whereas COP-1 carries cargo in a retrograde direction, ie, from the golgi to the ER

Front 62

Describe the 2 current models of how transporting vesicles from the ER to the golgi takes place.

Back 62

- the vesicular transport model believes that separate vesicles are sent from the ER to the golgi network, from there to the cis, from there to the medial, etc.
- the cisternal maturation model believes that separate vesicles are released from the ER to the golgi network, but once it fuses to the cis face of the golgi, all the vesicles fuse and simultaneously move through the golgi toward the trans face and then released and the vesicles reform (they move as a sheet through the golgi)

Front 63

How do the newly made proteins accumulate in particular transport vesicles?

Back 63

By the "sorting signal" on the protein

Front 64

How does the outer coat of a coatomer get released from the vesicle?

Back 64

Hydrolysis of GTP

Front 65

What are a t-snare and a v-snare?

Back 65

The v-snare is a vesicle protein on the outside that is complementary to a t-snare protein on the target organelle. This ensures that the vesicle goes to the right place.

Front 66

What is a Rab protein?

Back 66

It belongs to the GTP superfamily and helps with the docking and fusion of a transport vesicle to the appropriate target. It also governs the rate of trafficking.

Front 67

How do the v-snare and t-snare attach to each other?

Back 67

In a coiled-coil fashion. Once they are coiled around each other, they promote fusion of the two membranes. Botox cleaves a particular v-snare located on neurotransmitter vesicles at the neuromuscular synapse, thereby preventing vesicles carrying neurotransmitter to fuse with the surface membrane. Failure to release neurotransmitter causes muscular paralysis.

Front 68

Describe the 3 types of cytoskeletal fibers.

Back 68

- microfilaments – 8 nm wide, made of actin, 2 intertwined strands
- intermediate filaments – 10 nm wide, keratin fibers wound into thicker cables
- microtubules – 24 nm, made of α- and β-tubulin that makes a hollow tube, cilia

Front 69

Describe actin and how it polymerizes.

Back 69

Actin exists as a globular monomer called G-actin and as a filamentous polymer called F-actin, which is a linear twisted pair of chains of G-actin subunits. Each actin molecule contains a molecule of either ATP or ADP. Actin filaments exhibit polarity; the end at which the ATP-binding cleft of the terminal actin subunit is exposed to the surrounding solution is designated the (-) end, at the (+) end, the cleft contacts the neighboring actin subunit and is not exposed. The (+) end elongates 5-10 times faster than the (-) end. The turnover of actin filaments at the leading edge of some migrating cells probably occurs by a treadmilling effect, with subunits added to filaments near the leading edge of the cell and lost from the rear end.

Front 70

Describe the types of myosin.

Back 70

Myosin is an actin-binding protein, and there are types 1, 2, 5, 6 and 11. Type 1 is a monomer, the others are dimers, the “head” of the structure has an ATPase activity; the dimeric forms “walk” along an actin filament

Front 71

Describe myosin type 2.

Back 71

Type 2 was the first to be discovered, it has 6 polypeptide chains, 2 heavy, 2 regulatory light chains and 2 essential light chains. It functions in muscle contraction. The ATPase activity of the myosin head is very slow (release of hydrolysis products is rate-limiting) but is stimulated by F-actin. Chymotrypsin and papain cleave type 2 to leave the two myosin S1 heads which will still bind F-actin, these when bound to F-actin form “stacked arrowhead-like” structures, in which the points of the arrows point in the direction of the (-) end of the polymer; “barbed” ends are in the direction of (+) end of decorated actin filament.

Front 72

Describe a bipolar bundle of myosin type 2 (thick filament).

Back 72

All the tails come together and leave the heads pointing outward; two such bundles are attached via the tail end of the bundle; where the tails of the two bundles join is called the “bare zone”, the whole bundle is 325 nm long

Front 73

Describe a myoblast.

Back 73

It is a muscle precursor cell, several fuse together to make a multi-nucleated cell (becomes muscle fiber cell), the nuclei get pushed to the outside, actin and myosin fill the cytoplasm; there is no cell division after fusion to form the multinucleated muscle fiber cell

Front 74

Describe the organization of a muscle.

Back 74

Myofilaments (actin or myosin bundles) make myofibrils, which are wrapped around by lacelike sarcoplasmic reticulum; myofibrils fill the cytoplasm of the muscle fiber; bundles of muscle fibers form a muscle fascicle; bundles of fascicles makes a muscle.

Front 75

Describe the bands in the sarcomere.

Back 75

The A-band is dark and stands for anisotropic and is the myosin bipolar bundles (“thick filaments”) overlapped with actin filaments; the I-band is light and stands for isotropic and is the thin filaments, the I-band has a Z-line going down the center of it, (the very center of a sarcomere is called the M-line)

Front 76

Describe a muscle contraction.

Back 76

The S-1 heads of the myosin in the thick filaments walk along the actin filaments toward the (+) end, thereby pulling the Z-lines towards the center of the sarcomere.

Front 77

What molecule is used as spacers between the actin filaments?

Back 77

α-actinin

Front 78

What molecule is used as caps at the (-) end of the actin filaments?

Back 78

Tropomodulin

Front 79

Describe the mechanism for rigor mortis.

Back 79

“Stiffness of death”, after death, there is no more ATP left in the muscle, therefore there is no release of myosin heads from actin filaments, nor can Ca++ be pumped out of the cell as it leaks in.

Front 80

What serves as a reservoir of high energy phosphate during muscle work?

Back 80

Creatine-phosphate

Front 81

What two proteins are responsible for the calcium regulation of the muscle?

Back 81

Troponin and tropomyosin

Front 82

Describe the sliding filament theory.

Back 82

1. attached – without a nucleotide, myosin binds tightly to actin
2. released – ATP binding to the ATP-binding site on myosin head causes it to release from actin
3. cocked – when the binding site surrounds ATP, this causes the head to move 5 nm, this causes ATP to hydrolyze, but ADP and Pi stay bound to the active site
4. force-generating – weak binding of myosin to actin in the new position causes release of Pi which causes the “power stroke”, this is the myosin head regaining original shape, this causes the release of ADP
5. attached – the myosin head is now locked tightly to actin, it needs ATP to restart cycle

Front 83

What is the sarcoplasmic reticulum?

Back 83

A specialized ER in the muscle cell cytosol that acts as a reservoir of Ca2+

Front 84

What happens when a nerve impulse reaches a skeletal muscle?

Back 84

It causes the opening of voltage-gated Ca2+ channels in the SR. The release of Ca2+ into the cytosol triggers contraction. In skeletal muscle, the cytosolic Ca2+ concentration influences the interaction of troponin/tropomyosin proteins with actin thin filaments.

Front 85

Describe troponin and tropomyosin and what they do in muscles?

Back 85

TM is a ropelike molecule strung together head to tail along each actin thin filament. Troponin-C controls the position of TM on the surface of an actin filament. In the absence of CA2+, myosin can bind to a thin filament, but the TM-TN complex prevents myosin from sliding along the thin filament. Binding of Ca2+ ions to TN-C triggers a slight movement of TM that exposes the myosin-binding sites on actin. At high Ca2+ concentrations, the inhibition exerted by the TM-TN complex is relieved, and contraction occurs. (TM covers the myosin binding sites so it is not always contracting; when Ca2+ is high, troponin will allow TM to uncover myosin binding sites on actin, so contraction can occur.)

Front 86

Describe the mechanism of rigor mortis.

Back 86

Since there is no ATP production after death, Ca++ can’t be pumped our, and accumulates in the muscle; also, the muscle stays in the “attached” state - myosin is bound tightly to actin in the “rigor” configuration, waiting for an ATP to bind (but there is no ATP). Since all the muscles are in this locked config, they are in the contracted, stiff state. In a living muscle, this state is short-lived because the muscle will have ATP to continue the cycle.

Front 87

What carries a wave of depolarization into the muscle fiber?

Back 87

Transverse tubules

Front 88

How do action potentials trigger muscle contraction?

Back 88

1. AP (action potential) arrives at end of axon and triggers release of acetylcholine; acetyl choline binds to muscle receptors, which triggers an action potential wave over the surface of the muscle surface.
2. AP propagates through T tubules and stimulates the Ca2+ release channel on sarcoplasmic membrane
3. This causes SR to release Ca2+ into cytosol
4. Sarcomeres contract in response to high Ca2+ levels (Ca2+ binds to Troponin; Ca++-Troponin pulls Tropomyosin away, exposes myosin-binding sites on underlying actin filament; myosin heads walk along actin filament, pulling it towards center of sarcomere.)
5. ATP is used to pump Ca2+ back into the SR

Front 89

What makes the “muscle triad”?

Back 89

Two SRs and a transverse tubule

Front 90

How does Ca2+ regulate smooth muscle?

Back 90

Ca2+ bind to calmodulin, which in turn activates myosin light chain kinase which phosphorylates individual myosin molecules, this permits them to assemble to form bipolar bundles. When the light chain is unphosphorylated, myosin 2 is inactive (unable to form bipolar bundles). Since this mode relies on the the action of protein kinases, it is slower than in skeletal muscle.

Front 91

Compare actin and myosin in skeletal and smooth muscle.

Back 91

In skeletal muscle, contraction is regulated by cycling of actin between on and off states. In smooth, contraction is regulated by cycling myosin 2 between on and off states.