Mcb130 Midterm I Flashcards

Front 1

Lecture 1. Each type of organelle has a characteristic ______ and _______ and therefore does what?

Back 1

1) Structure.
2) Molecular composition.
-Carries out a specific task.

Front 2

Lecture 1. Assembly of ribosomal subunits occurs where?

Back 2

In the nucleus.

Front 3

Lecture 1. Membrane topology. The interior of the vesicle is topologically equivalent to what leaflet of the PM? What about the nuclear envelope?

Back 3

The interior of the vesicle and the inner nuclear envelope are equivalent to the outer leaflet of the PM and previously faced the exterior of the cell.

Front 4

Lecture 1. Compartmentalization requires the expenditure of what?

Back 4

Energy in the form of GTP, ATP.

Front 5

Lecture 1. The five advantages of compartmentalization?

Back 5

1) Allows the cell to achieve high local concentrations.
2) A way of increasing the surface area of the cell with large volume.
3) Maintaining different local micro-environments.
4) Sequestration of potentially harmful chemicals.
5) A way of providing additional layers of regulation.

Front 6

Lecture 1. Proteins use what to get to their appropriate locations?

Most of the time, proteins may have _________ to facilitate the various stages of their transport to a final destination.

Back 6

Proteins use signal sequences to get to their appropriate places. These signal sequences can be one continuous stretch of amino acids or formation of a structure generated by folding different parts of the protein.

Proteins may have multiple signal sequences to facilitate in the different stages of transport to their final location.

Front 7

Lecture 2. Proteins can be transported to their final locations via what three ways?

Back 7

1) Gated transport-proteins are synthesized and folded in the cytosol and go to the nucleus and come back out. The protein has to traverse the nuclear pore in its folded state.
2) Trans-membrane- proteins cross the lipid bilayer in a folded state.
3) Vesicular transport- transport in vesicles.

Front 8

Lecture 2. It is the ________ that gives the nucleus structure and function and breaks down during mitosis.

Back 8

Nuclear lamina.

Front 9

Lecture 2. The nuclear pore complexes can allow transport of smaller molecules. For larger molecules, the NPC requires_________.

Back 9

The binding of the cargo to specific receptor proteins that can interact with the NPC to facilitate their transport through the pore.

Front 10

Lecture 2. Ribosomal proteins are made in the _______. rRNAs of ribosomes are made in the _______. Ribosomal subunit assembly occurs in the _______, then moved where?

Back 10

Ribosomal proteins are made in the cytoplasm. Then, it is imported into the nucleolus to be combined with rRNAs (which are made in the nucleus) to form the ribosomal subunits. The subunits are exported to the cytosol to be made into ribosomes.

Front 11

Lecture 2. Is the nucleolus surrounded by membranes?

Back 11

No.

Front 12

Lecture 2. The nucleoporins, namely the FG repeats, serve what function?

Back 12

Preventing large molecules from diffusing into the nucleus.

Front 13

Lecture 2. Using biochemical studies of nuclear transport: digitonin permeabilized cells. See the importance of in vitro and in vivo studies in certain biological processes. There is a parallel in studying protein translocation into Er using liposomes (in vitro) and microsomes (in vivo).

Back 13

1) Reconstituted nuclear transport in vitro.
2) Add fluorescently labeled proteins that have SV40 NLS = no transport.
3) Fluorescent labeled proteins plus cytoplasmic lysate = transport.
4) Found that importin alpha and importin beta and Ran in the cytoplasmic lysate were necessary for nuclear transport.

Front 14

Lecture 2. Usually, importin alpha and importin beta form a _______. Importin alpha binds to _____ and importin beta binds to _______.

Back 14

-Importin alpha and beta form a heterodimer.
-Importin alpha binds to the cargo and importin beta binds to the NPS components (the FG repeats).

Front 15

Ran-GEF is localized in the _______ and Ran-GAP is localized in the _______. Therefore, most of the Ran in the cytoplasm is in the GDP/GTP bound form. What about in the nucleus?

Back 15

Ran-GEF is localized in the nucleus and Ran-GAP is localized in the cytoplasm. Hence, most of the Ran in the nucleus is in the Ran-GTP and in the cytoplasm it is in the Ran-GDP form.

Front 16

Lecture 2. It is the differential concentrations of Ran-GTP made possible by the different locations of ______ and ______ that makes nucleo-cytoplasmic transport possible.

Back 16

Ran-GAP and Ran-GEF.

Front 17

Lecture 2. A full cycle of transporting a cargo molecule into the nucleus takes _______ and you get back ________ and ______.

Back 17

1 GTP and get back a free Ran and importin.

Front 18

Lecture 2. Outline the steps taken by an entire cycle of transporting a cargo molecule into the nucleus.

Back 18

1) Cargo binds to importin.
2) Cargo with importin, along with Ran-GDP, diffuses down a concentration gradient into the nucleus.
3) Ran-GEF in the nucleus makes the conversion from Ran-GDP to Ran-GTP, which drives step 4.
4) Cargo dissociates with the importin, and importin binds with the Ran-GTP.
5) Importin-RanGTP complex diffuses down a concentration gradient out to the cytoplasm and Ran-GAP forces the conversion of Ran-GTP to Ran-GDP, which causes the complex to dissociate.
6) Importin is free to bind to cargo again.

Front 19

Lecture 2. Outline the cycle of the Ran dependent export of cargo from the nucleus.

Back 19

1) Nuclear exportin+cargo+Ran-GTP in a trimer are exported from the nucleus through the NPC.
2) Ran-GAP causes the conversion of Ran-GTP to Ran-GDP
3) Conversion forces the dissociation of cargo+exportin+Ran-GDP.
4) Ran-GDP (which is changed into Ran-GTP upon entry into nucleus by Ran-GEF) and exportin can freely diffuse into the nucleus again to repeat cycle.

Front 20

Lecture 2. T or F. The nuclear export sequences can both be on the same cargo.

Back 20

True. This gives the phenomenon of shuttling.

Front 21

Lecture 2. mRNA is exported from the nucleus via a Ran independent or Ran dependent pathway?

Back 21

Ran independent.

Front 22

Lecture 2. Describe a full cycle that involve the export of mRNA out of the nucleus.

Back 22

1) After transcription of DNA and all necessary post-transcriptional modifications (poly A tail, the cap at beginning, and transport proteins) the mRNA is able to engage the FG repeats on the NPC and be exported to the cytoplasm.
2) Transport proteins dissociate and diffuse back into the nucleus to repeat the process again.

-People are still unsure as to where the energy source needed to create the gradient comes from.

Front 23

Lecture 2. How is the restoration of nuclear localization of proteins after mitosis achieved?

Back 23

1) Ran-GEF is associated with the chromosomes during mitosis. (shown by florescent probes)
2) Ran-GAP is excluded from this localization.
3) When the nuclear membrane forms again there is a gradient of Ran-GTP between the nucleus and cytoplasm which allows nucleo-cytoplasmic transport.

Front 24

Lecture 3. What are the three types of lipids found in animal cell membranes?

Back 24

1) Phosphoglycerols-glycerol backbone.
2) Sphingolipids-sphingosine backbone.
3) Cholesterol (% content can be as high as 30%)

Front 25

Which organisms cell membranes lack cholesterol?

Back 25

Plants and bacteria.

Front 26

Lecture 3. Regardless of whether you are dealing with saturated or unsaturated fatty acids, there must always be one fatty acid chain that's _____________.

Back 26

There must be one fatty acid chain that's completely saturated.

Front 27

Lecture 3. In phospholipids, what two things can confer different biochemical properties?

Back 27

Different head groups and fatty acid tails.

Front 28

Lecture 3. What kind of phospholipids exhibits a amide linkage instead of an ester linkage between the backbone and the fatty acid tail(s)?

Back 28

Sphingolipids.

Front 29

Lecture 3. Do all cell membranes have the same composition of phosphoglycerides and sphingolipids?

Back 29

No.

Front 30

Lecture 3. Phosphotidyl-ethanolamine is important for what?

Back 30

It is important for the activation of certain enzymes in the inner leaflet of the plasma membrane.

Front 31

Lecture 3. Phosphotidylserine is seen in which leaflet of the PM? What about phosphotidylcholine?

Back 31

Phosphotidylcholine-outer leaflet.
Phosphotidylserine-inner leaflet.

Front 32

Lecture 3. Sphingomyelin is seen most abundantly in the ______.

Back 32

Brain.

Front 33

Lecture 3. Cholesterol is characterized by three main regions. What are they?

Back 33

1) A polar hydroxy head group.
2) A rigid ring structure.
3) A nonpolar hydrocarbon tail.

Front 34

Lecture 3. What are the two type of artificially constructed membranes discussed in class?

Back 34

1) Black membranes.
2) Liposomes.

Front 35

Lecture 3. Membrane fluidity/viscosity varies based on three factors, which are __________, _____________, and ____________.

Back 35

1) Length of acyl chain.
2) Amount of saturation.
3) Temperature.

Front 36

Lecture 3. Cholesterol acts as a __________ to membrane fluidity with temperature.

Back 36

Buffer.

Front 37

Lecture 4. Flipase is what type of transporter?

Back 37

ABC.

Front 38

Lecture 4. Peripheral proteins are bound to the membrane via ________ interactions to the _________ and _________.

Back 38

Electrostatic.
Phospholipids and other proteins.

Front 39

Lecture 4. SDS is a charged/non-charged detergent and penetrate the interior of the protein via ________ and can solubilize and denature the hydrophobic interior of the protein.

Back 39

SDS is an anionic detergent and can interact with both the hyphobic and hydrophilic regions of the protein being extracted. It penetrates the interior of the protein via interactions with the hydrophilic side chains.

Front 40

Lecture 4. Do non-ionic detergents like Triton-X have amphipathic character? Do they denature proteins?

Back 40

Yes. No.

Front 41

Lecture 4. Detergents form micelles at the _____________.

Back 41

CMC.

Front 42

Lecture 4. When one chooses a proper detergent to extract membrane protein, one needs to choose the detergent that's __________________.

Back 42

Need a detergent that has the lowest concentration needed to get desired effect and one that doesn't disrupt the protein's functionality.

Front 43

Lecture 4. Detergents interfere with the _______ that are used for structural studies and do what else?

Back 43

Crystallization by interfering with the packing of the proteins into a crystalline structure.

In addition, detergents may interfere with functional assays as they are so packed into the proteins

Front 44

Lecture 4. Describe detergents and its substrates before and after the extraction process.

Back 44

-Before: detergent monomers equilibrate with their micelle counterparts and begin to engage the lipid bilayer.
-After: detergent monomers are seen coating the hydrophobic parts of the trans-membrane protein and phospholipids.

Front 45

Lecture 4. After reconstitution, the function of the protein can be studied if _______________. This is usually not the case because reconstitution usually results in ______________, in contrast with the ___________ seen in normal cell membranes.

Back 45

If the protein is inserted in the correct orientation.
Mixed topology.
Inherent asymmetry.

Front 46

Lecture 4. Can use _______ to look for likely trans-membrane amino acid sequences in the trans-membrane domain.

Back 46

Bio-informatics.

Front 47

Lecture 4.In the alpha helical conformation of each trans-membrane domain of trans-membrane proteins, the ______ of each amino acids forms a hydrogen bond with the __________ group of the amino acid that is where.

Back 47

CO group of each aa form a h bond with the NH group of the aa that is 4 aa c-terminal to it.

Front 48

Lecture 4. Usually there are two types of disulfide bonds found in the extracellular domains of transmembrane proteins.

Back 48

Intra-chain and inter-chain disulfide bonds.

Front 49

Lecture 4. Disulfide bonds and sugar groups on proteins are forms in the ______ and ___________, so found in the outside of the membrane.

Back 49

ER and Golgi.

Front 50

Lecture 4. The cytosol usually has a reducing/oxidative environment. What about the extracellular matrix? That is why you see disulfide bonds in the extracellular environment and _________ in the cytosol?

Back 50

Cytosol has a reducing environment. Extracellular domain has an oxidizing environment.
Sulfhydryl groups.

Front 51

Lecture 5. Other than hydrophobic molecules, only ________ and ___________ can freely diffuse across a lipid bilayer.

Back 51

Gases and small uncharged polar molecules.

Front 52

Lecture 5. In facilitated diffusion, the channel can be gated or non-gated, which responds to a particular signal (such as voltage). You can alter the direction of transport by _____.

Back 52

FYI. Altering the direction of the gradients (electrical, chemical, electrochemical)

Front 53

Lecture 5. Often, facilitated diffusion usually deals with _________ diffusing down ______ gradients.

Back 53

Ion. Electrochemical gradients.

Front 54

Lecture 5. Facilitated diffusion dealing with other (mostly non-ionic) molecules involves a transporter that transports molecules via conformational changes called ______

Back 54

Uniporter.

Front 55

Lecture 5. Symporters and anitporters use energy from ___________ to drive the transport molecules UP their gradient. This process is called ________. Transport usually involves a conformational change in the transporter.

Back 55

Pre-existing electrochemical gradients. Secondary active transport.

Front 56

Lecture 5. Are Na+/K+ ATPase secondary active transport proteins?

Back 56

No. They require ATP to transport Na+ out of the cell and K+ inside the cell.

Front 57

Lecture 5. In facilitated diffusion, the channel can be gated or non-gated, which responds to a particular signal (such as voltage)

Back 57

FYI.

Front 58

Lecture 5. Often, facilitated diffusion usually deals with _________ diffusing down ______ gradients.

Back 58

Ion. Electrochemical gradients.

Front 59

Lecture 5. Secondary active transport dealing with other (mostly non-ionic) molecules involves a transporter that transports molecules via conformational changes called ______

Back 59

Uniporter. Yes, even uni-porters use secondary active transport by relying on pre-existing gradients.

Front 60

Lecture 5. Symporters and anitporters use energy from ___________ to drive the transport molecules UP their gradient. This process is called ________. Transport usually involves a conformational change in the transporter.

Back 60

Pre-existing electrochemical gradients. Secondary active transport.

Front 61

Lecture 5. Are Na+/K+ ATPase secondary active transport proteins?

Back 61

No. They require ATP to transport Na+ out of the cell and K+ inside the cell.

Front 62

Lecture 5. Describe how the GLUT I transporter works.

Back 62

Glucose binds to extracellular domain of the transporter, which triggering a conformational change that lets it diffuse down the gradient into the cell. GLUT I returns to its original conformation and affinities. The glucose is immediately phosphorylated so free glucose inside cell is low and maintains gradient. Phosphorylated glucose cannot rebind to the transporter.

Front 63

Lecture 5. What are the four types o transporters that use ATP hydrolysis to pump molecules?

Back 63

1) P class- Na+/K+ ATPases.
2) V-class- proton pumps.
3) F-class- proton pumps in reverse direction, sometimes giving ATP production. (inner mitochondrial membrane).
5) ABC transporters- transport of a variety of molecules.

Front 64

Lecture 5. Describe how a P class pump works.
(e.g. a Ca++ pump) Also describe why the Ca++ pumps are well understood by scientists.

Back 64

1) E1 conformation-2 high affinity biding sites on the cytosolic side.
2) ATP hydrolysis and phosphate put on aspartate residue.
3) Phosphorylation causes a conformational change that causes a change in affinities (low affinity for Ca++ in the cytosolic side) and causes the release of Ca++ to the outside of cell (E2).
4) Dephosphorylation returns the pump to its original affinities and conformation (E1).
-Ca++ pumps easily purified because it comprises 80% of the proteins on the membrane of the sarcoplasmic reticulum.

Front 65

Lecture 5. How does the conformational change from E1 to E2 in the calcium pump release the Ca++ from the pump into the SR lumen?

Back 65

The helices providing the ionic interactions in the pump move apart and release the Ca++ in the E2 state, which is when the phosphate is still bound to the pump.

Front 66

Lecture 5. In the Na+/K+ ATPase, both Na+ and K+ are pumped with or against their gradient? How many Na+s moved and how many K+s moved?

Back 66

Against gradients. 3 Na+s moved and 2 K+s moved per cycle.

Front 67

Lecture 5. Describe the full cycle of how the Na+/K+ ATPase pump works.

Back 67

1) E1 conformation = pump has high affinity for Na+ binding. ATP binds to pump.
2) Phosphorylation of aspartate residue on pump.
3) Pumps changes conformation from E1 to E2 (high affinity for K+ binding), during which K+s move into pump and Na+s move out of pump.
4) Dephosphorylation (E2 state makes this favorable) and release of K+ and return to original E1 conformation.

Front 68

Lecture 5. The V1 domain of V class pumps resides in the _________. What about the V0 domain?

Back 68

Cytoplasm. The V0 domain is in the transmembrane.

Front 69

Lecture 5. What causes the protons to move through the channels set up by subunits of the V0 domain of the V class pumps?

Back 69

ATP hydrolysis of the V1 domain.

Front 70

Lecture 5. How is there no electrical potential or charge buildup between the membranes of the V class pump as protons are pumped into the inner space?

Back 70

There are chloride channels that allow free diffusion of chloride into inner space. This, however, does not kill the concentration gradient.

Front 71

Lecture 5. What are two fundamental differences between pumps and channels?

Back 71

Pumps never allow a molecule access to both sides of a membrane at the same time.
Pumps works slower.

Front 72

Lecture 5. Describe how the F class pumps work.

Back 72

There is a subunit that either synthesizes or hydrolyzes ATP depending on direction that the pump works in.
Other subunit is a channel allowing the movement of protons, which causes the rotation of the rings at other end of the pump, forcing the production of 1 ATP ever 1/3 rotation of the ring.

Front 73

Lecture 6. What are the differences seen in the shape of the rough and smooth ER?

Back 73

Smooth ER has no ribosomes and is tubular in shape while the rough ER does have ribosomes and is sheet like in shape.

Front 74

Lecture 6. What were the original ideas behind the signal sequence hypothesis? What's the point of having tight pores in the ER?

Back 74

Scientists thought that just adding mRNA (with a signal sequence) and ribosomes would be enough for the co-translation of the protein, as the pore on the ER would be recognized by the signal sequence. After translation, the signal sequences would be cut by a peptidase. This is not the case, as they found.

-There are different concentrations between the ER lumen and outside.

Front 75

Lecture 6. What is SRP made out of?

Back 75

6 proteins and 1 RNA (thought to function by holding the proteins together).

Front 76

Lecture 6. What causes the stopping of arrest of translation caused by SRP?

Back 76

The dissociation of the SPR from its receptor.

Front 77

Lecture 6. What does SRP do to translation by the free ribosome?

Back 77

Once the first hydrophobic stretch (most likely the signal sequence) is translated, SRP (with a hydrophobic groove) binds to signal peptide and stops translation.

Front 78

Lecture 7. The purified sec 61 channel has 3 subunits which are _____, _______, and ___________. Which subunit is actually the channel?

Back 78

Alpha, beta, and gamma. Alpha

Front 79

Lecture 7. What ultimately convinced scientists that this Sec 61 channel was the last thing they needed to achieve sufficiency of protein translocation into the ER?

Back 79

A reconstitution experiment where inclusion of the sec61 channel into liposomes and everything else added beforehand was enough to get protein translocation.

Front 80

Lecture 7. There are how many ways the Sec 61 channel can open?

Back 80

2.

Front 81

Lecture 7. How does the incoming protein/SRP complex even begin to engage the Sec61 channel to begin translocation?

Back 81

The SRP/SRP receptor complex can interact favorably with the Sec61 channel to allow the entire complex to dock to.

Front 82

Lecture 7. Describe the process in which a soluble, ER bound protein is translocated through the ER membrane.

Back 82

1) SRP/ribosome complex is presented to the SRP receptor on ER surface.
2/3) The signal sequence at the N terminus is presented to the channel in a curved fashion.
2/3) SRP dissociated from its receptor and translation begins.
4) Signal peptidase cleaves off the signal sequence, which diffuses laterally throughout the ER membrane to be degraded later.
5) Protein now successfully translocated into ER lumen.

Front 83

Lecture 7. Scientists think that the energy needed for protein translocation into the ER comes from _____________.

Back 83

The ribosome.

Front 84

Lecture 7. Where does the N and C terminus face in the Type I transmembrane protein?

Back 84

C terminus is in the cytosol and the N terminus is in the ER lumen.

Front 85

Lecture 7. How is the T1 transmembrane protein threaded through the ER membrane.

Back 85

Same thing as translocating a soluble protein but the protein has an additional hydrophobic region that acts as a stop sequence which stops the translocation process and engages a hydrophobic part of the channel m allowing it to pry open the Sec61 channel, which allows it to diffuse out and stay in the ER membrane.

Front 86

Lecture 7. What would happen if you got rid of all the stop sequences on a Type I transmembrane protein?

Back 86

It would be secreted.

Front 87

Lecture 7. What is the difference between T2 and T3 transmembrane proteins in the sequence of their amino acids? How does this affect how they're placed on the ER membrane?

Back 87

In T2, positively charged amino acids are distributed near the N terminus, negatively charged amino acids are distributed near the C terminus, and vice versa for T3 transmembrane proteins. This distribution of charged affects how the ribosome/SRP complex is presented to the ER membrane, which then affects the orientation in which the protein is threaded through. Positively charged AAs will always remain in the cytosol and negatively charged AAs will always face the ER lumen.

Front 88

Lecture 7. Which type of transmembrane proteins has a terminal signal sequence? How does the stop transfer sequence stop the translation?

Back 88

Only Type 1.

-The stop transfer sequence engages a hydrophobic part of the bilayer and the channel opens and the protein is let out.

Front 89

Lecture 7. Which side of the ER membrane (lumen or cytosol) does the N terminus face in T2 transmembrane proteins? What about Type 3?

Back 89

-T2: N terminus is facing cytosol and C terminus is facing lumen.

-T3: N terminus is facing lumen and C terminus is facing cytosol.

Front 90

Lecture 7. In Type IV transmembrane proteins, SRP engages the protein when?

Back 90

Everytime there is a start sequence.

Front 91

Lecture 7. What determines what the ER membrane would look like with the transmembrane protein in it? What would determine the topology of the other terminus of the transmembrane protein?

Back 91

The initial transmembrane protein. Whether the transmembrane protein has an odd or even number of transmembrane domains.

Front 92

Lecture 7. What are the two types of post-translational translocation discussed in class?

Back 92

1) Translocation occurring via BIP, which provides energy for transport from ATP hydrolysis. Multiple Sec proteins form a complex which unfolds and threads translocating proteins in and the BIP prevents the proteins from going back out.
2) A Sec A protein unfolds ad threads the protein in using ATP.

Front 93

Lecture 7. The initial transmembrane domain of a Type 4 transmembrane protein is determined by what?

Back 93

By the same way that the orientation of Type 2 and 3 transmembrane proteins are detemined (charge distribution).

Front 94

Lecture 7. In a protein with an N terminus signal sequence, where does the N terminus reside with respect to the ER membrane?

Back 94

In the ER lumen.

Front 95

Lecture 7. If the starting transmembrane domain (start sequence) is a type transmembrane protein in which the N terminus is facing the lumen, there MUST be _________________.

Back 95

Another start sequence immediately beside the first transmembrane domain.

Front 96

Lecture 7. Where is the asymmetry of the transmembrane (esp. multipass ones) created?

Back 96

The threading of the transmembrane protein during translocation.

Front 97

Lecture 8. What are the 5 modifications of proteins that occurs in the ER and Golgi?

Back 97

1) Glycosylation.
2) Formation of disulfide bonds.
3) Folding f polypeptide bonds.
4) Assembly of multi-subunit complexes.
5) Proteolytic cleavage.

Front 98

Lecture 8. The extracellular domains of proteins is stabilized by what bonds? Are these bonds all created in the same order or not?

Back 98

Disulfide bonds, which are all created in the same way but modified in the ER and later in the golgi.

Front 99

Lecture 8. Why are only the extracellular domains of glycoproteins glycosylated?

Back 99

Because glycosylation only occurs in the ER lumen which occurs right after the protein is translocated.

Front 100

Lecture 8. What is the amino acid sequence (motif) needed for glycosylation?

Back 100

Asn-X-Ser/Thr, with X being any AA except proline.

Front 101

Lecture 8. Is the Asn-X-Ser/Thr motif itself sufficient for glycosylation?

Back 101

No. It is necessary but the motif needs to be in the ER lumen.

Front 102

Lecture 8. The oligosaccharides that are to be transferred to a glycosylated protein are initially made where, what enzyme is needed to transfer the oligosaccharide sequence to the protein? When is the oligosccharide transferred to the protein?

Back 102

Dilochiol. Need a oilgosaccaryl transferase for the transfer. When it is being co-translated into the lumen of the ER.

Front 103

Lecture 8. Synthesis of the oligosaccaride precursor later used for protein glycosylation is done where? What happens to it afterwards?

Back 103

The synthesis of the oligosaccharide precursor occurs in the cytosol (because the enzymes needed for this are in the cytosol) but is flipped to the other side after.

Front 104

Lecture 8. What retains incompletely folded proteins in the ER?

Back 104

Calnexin.

Front 105

Lecture 8. What 2 processes are needed for proteins to exit the ER? What does this ensure?

Back 105

Normal protein folding and glucose trimming. This guarantees quality control when the two processes are linked.

Front 106

Lecture 8. Name the stops that an an unfolded protein has to go through from the very beginning (with three glucoses rather than just one) to the very end when it is transported out of the ER.

Back 106

1) Protein has two of its three glucose trimmed off.
2) The protein engages calnexin, a chaperone, which keeps it in the ER.
3) Removal of terminal glucose by glucosidase removed protein from calnexin (a membrane bound chaperon), and protein engages glucosyl transferase.
4) Glucosyl transferase determines whether protein is folded correctly or not. If not, a glucose is added to it so it can re-engage calnexin so it stays in the ER.
-This cycle continues to occur until protein has all glucose trimmed off and has folded properly.
-You need both folded protein and glucose trimming to exit ER lumen because that ensures complete, proper folding of protein before its exit.

-Just one glucose is enough to engage the calnexin

Front 107

Lecture 8. How are unfolded proteins exported from the ER and degraded?

Back 107

A slow mannose marks the proteins that have been in the ER for a long time (because of improper folding).

These proteins are then stripped of their sugars (by glycenase) and moved back into the cytosol via the Sec61 pore and degraded by proteosomes (by being tagged with ubiquitin)

Front 108

Lecture 8. What are the three main "unfolded protein response" that promotes the synthesis of chaperones that function in the ER?
What happens when there are too many unfolded proteins in the ER?
Where are these sensors for unfolded proteins?

Back 108

1) Regulated mRNA splicing to initiate translation of gene regulatory proteins.
2) Phosphorylation of sensor blocks the translation of all proteins except chaperone proteins.
3) ATF6 sensor is cleaved and becomes the transcription factor to activate genes that function as chaperones.



-The secretory pathway may be clogged if too many unfolded proteins reside in the ER.
-Sensors are on the ER with action surface on the cytosol surface.

Front 109

Lecture 8. Describe in detail the first pathway involving mRNA splicing that the cell has against too many unfolded proteins in the ER.

Back 109

In the ER, an unfolded protein activates BIP, which then activates Ire1 to dimerize. The Ire1 becomes an endnuclease, which removes an mRNA segment, which forms an RNA segment capable of being translated into a transcription factor called Hat1, which enhances translation of chaperone genes.

Front 110

Lecture 8. What is the role behind adding that one glucose to unfolded proteins in the ER? What's the point of cutting off that final glucose after it has gone through calnexin?

Back 110

So it can return to th calnexin for another round of attempted proper folding.
So it can go to the enzyme and get another glucose added to return to calnexin. If it is already folded, it will exit the ER---> it is that terminal glucose that makes the connetion between calnexin and the checking enzyme.

Front 111

Lecture 9. Membrane movement between two compartments occurs with _______. In this process, the ______ is preserved, _______ fuse with highly specific _____, and vesicles are highly enriched in ________.

Back 111

Vesicular trafficking.

-Topology, vesicles fuse with specific target compartments.

-Vesicles are highly enriched in components of the donor compartment.

Front 112

Lecture 9. What two things do coat proteins do to vesicles?

Back 112

1) Incorporation of specific cargos.
2) Curvature of membrane to form vesicles.

Front 113

Lecture 9. Describe the sequence of events that occur for a cargo to be transported from the ER to golgi.

Back 113

1) Sar1-GDP is activated to Sar1-GTP.
2) Sar1-GTP extends its ampipathic tail and becomes embedded into the membrane.
3) The Sar1-GTP recruits Sec 23/24, which recruit cargo molecules that display sorting signals.
4) Sec 23/24 also recruits Sec 31/13 that comprises the outer coat.
5) Coat disassembly occurs, which requires GTP hydrolysis.

Front 114

Lecture 9. What does the curvature of the membrane brought about Sec 13/31 do for vesicular trafficking?

Back 114

The curvature of the member brought about the polymerization of Sec31/13 causes a increase of the local concentrations of cargo.

Front 115

Lecture 9. What is used by vesicles and the target compartment to ensure that only specific vesicles fuse with specific target compartments?

Back 115

1) Specific pairs of v and t snairs.
2) Rab proteins with binds with specific effectors.

Front 116

Lecture 9. Which regulatory domain acts first when a vesicle is about to fuse with its target compartment? Rab proteins binding to their effectors or v and t snares binding? What happens after that?

Back 116

Rab proteins binding to their effectors. The v and t snares fuse, which squeezes out all the water in between the two compartments and initiates membrane fusion.

Front 117

Lecture 9. What cell model is said to be a great way to study vesicle fusion? The v snares are usually on the target or donor compartment? What about t snares?

Back 117

Synaptic vesicles. The v snares are usually on the donor compartment membrane and t snares are on the target compartment.

Front 118

Lecture 9. What do neurotoxins and clostridium tetani and butuli do to the snares?

Back 118

They act as proteases that cleave these v and t snares, essentially stopping vesicle fusion and neural transmission.

Front 119

Lecture 9. Continued vesicular trafficking using one type of vesicle needs _________ (a protein) in addition to specificity conferred by Rab proteins and the v and t snares. What happens if there is a mutation in this protein?

Back 119

NSF proteins, which use energy from ATP to pry apart the v and t snares after they have fused. If there is a mutation in this protein, vesicles from the ER will accumulate and cannot fuse with Golgi. Also, new vesicles cannot be made as old vesicles will have their v snares tied up with t snares, without being recycled for new vesicles.

Front 120

Lecture 9. How are ER resident proteins that are accidentally transported to the Golgi retrieved?

Back 120

Soluble resident ER proteins will have a C terminus KDEL motif that allows it to bind to a cop-I coated vesicle back to the ER by binding to the KDEL receptor in the vesicular tubular cluster in the golgi. Transmembrane proteins that resident in the ER do the same thing, but they have a different motif (KKXX) on N terminus, which is cytosolic and binds to COPI subunits.

Front 121

Lecture 9. How can one tell that an ER protein has been accidentally transported to the golgi and transported back? How to prove that retrograde transport exists.

Back 121

There are enzymes that ONLY reside in the golgi (e.g. mannosidase I) that might have acted on the ER resident proteins before being transported back. This modification can be detected quite easily.

Front 122

Lecture 9. How are the soluble ER proteins able to associate with the KDEL receptors in the golgi and dissociate with the same receptor in the ER?

Back 122

In the Golgi, which has a lower ph, ER resident proteins with a KDEL motif are encouraged to bind to the KDEL receptor. When the complex gets to the ER, which has a higher ph, the cargo is encouraged to dissociate.

Front 123

Lecture 9. Is it possible to get a vesicle without cargo in a live cell? In vitro?

Back 123

In vitro-yes.
In vivo-no.

Front 124

Lecture 9. Golgi enzymes are usually transmembrane/soluble proteins with their active sites where?

Back 124

Transmembrane proteins with their active sites in the lumen.

Front 125

Lecture 9. Golgi enzymes must ALL have passed through where, and must have _______ to retain itself in a certain compartment of the golgi.

Back 125

ER. Signal sequences.

Front 126

Lecture 9. What are the two models that describe how stuff moves through the golgi?

Back 126

1) Cisternal maturation-the vesicular tubular structures aggregate and form the cis golgi compartment, which moves and gradually mature into further compartments, stuff in each compartment don't move, but the enzymes move backwards in retrograde transport via cop I vesicles to previous stacks.
2) The comparments of the golgi are static and vesicles carrying the cargo move forward from one stack to the next.

Front 127

Lecture 9. What's wrong with the old model of cargo movement in the golgi?

Back 127

There are large aggregates of cargo which are seen to pass through the golgi without having to be transported in a vesicle. Also, these cargo complexes are too big to fit inside vesicles.

Front 128

Lecture 9. Can cargo that's to be moved back to the ER move directly from the medial or trans golgi to the ER?

Back 128

No, must move to the cis cistern of the golgi first.

Front 129

Lecture 9. What is the vesicular tubular cluster?

Back 129

Where clusters of vesicles coming from the ER all fuse together before engaging the golgi.

Front 130

Lecture 10. What are the three ways types of compartments in the cell that can mature into a lysosome?

Back 130

1) Phagosome formed from phagocytosis.
2) Endosomes formed from endocytosis.
3) Autpphagosomes formed from autophagocytosis.

Front 131

Lecture 10. Outline the steps hydrolases go from the ER to the lysosomes.

Back 131

1) Lysosomal hydrolases are translocated into the ER and in the process is N-link glycosylated.
2) The glycosylated hydrolases bind to the GlcNac phosphotransferases in the ER lumen after the enzyme recognizes the signal patch on the hydrolase (2 separate patches that are together sufficient but necessary individually).
3) The phosphotransferase also binds to UDP-GlcNac.
4) The transferase transfers the GlcNac-P to the mannose catalytic site on the hydrolase.
5) The hydrolase is released, then sheds off the GlcNac, giving the M6P.
6) M6P binds to M6P receptors in the trans golgi) which can bind to clathrin, which polymerizes and forms a clathrin coated vesicle which leaves and fuses with late endosomes.
7) The M6P is let go of the hydrolases in the acidic environment. The phosphate is removed by acid phosphatases. The Phosphate is removed so mannose doesn't recombine with the hydrolase.
8) Hydrolases are activated by the acidic environment.

Front 132

Lecture 10. Where does the sorting of lysosomal proteins occur?

Back 132

In the trans-golgi.

Front 133

Lecture 10. After the mannose 6 phosphate receptor lets go of the mannose 6 phosphate in the lysosome, where does it go?

Back 133

It is recycled by being transported back to the golgi by being incorporated into retromer coated vesicles.

Front 134

Lecture 10. What happens to lysosomal hydrolases that accidentally escape to the exterior cellular environment? Outline all the steps.

Back 134

Some hydrolases, which are incorporate into clathrin coated vesicles, can exit the cell via exocytosis.

1) There are mannose 6 phosphate receptors on the cell surface, which captures the enzymes form aggregates that turn into clathrin coated vesicles used for endocytosis.
2) The vesicle sheds the clathrin and fuses with late endosomes.
3) The M6P receptors are recycles to either the trans golgi or PM.

Front 135

Lecture 12. True or false. Cholesterol can be made by the body as well.

Back 135

T.

Front 136

Lecture 12. What are the three main functions of cholesterol for the cell?

Back 136

1) Cell communication .
2) Membrane fluidity buffer with temperature.
3) Precursors to many biologically important molecules.

Front 137

Lecture 12. How is the HMG-CoA reductase regulated?

Back 137

Feedback (negative) inhibition.

Front 138

Lecture 12. Outline the steps of the cholesterol biosynthetic pathway.

Back 138

1) Acetyl-CoA combines with acetoacetyl Co-A to form HMG Co-A.
2) HMG Co-A reductase turns HMG Co-A into mavelonate.
3) Mavalonate turns into IPP.
4) IPP turns into squalene, which becomes cholesterol.

Front 139

Lecture 12. What is the limiting component of the cholesterol biosynthetic pathway?

Back 139

HMG-Co-A reductase.

Front 140

Lecture 12. How does the cell deal with high cholesterol in terms of dealing with HMG Co-A reductase?

Back 140

The cell stops cholesterol synthesis by degrading existing HMG Co-A reductase and also suppresses the gene transcription of the enzyme.

Front 141

Lecture 12. The HMG Co-A reductase enzyme has its active site where? How many trans-membrane domains does it have and how many are used to sense cholesterol levels? How is it degraded?

Back 141

The HMG Co-A reductase enzyme has 8 transmembrane domains, and uses 5 of them to sense cholesterol levels. Binding of cholesterol to the 5 domains causes the ubiquidation of the enzyme, which leads to its degredation.

Front 142

Lecture 12. How is cholesterol transported in out bloodstream?

Back 142

LDL particles.

Front 143

Lecture 12. What does the apoB lipoprotein do for LDL?

Back 143

Allows it to bing to LDL receptors.

Front 144

Lecture 12. Cholesterol looks like what while inside the LDL?

Back 144

Cholesterol travels in its ester form, which means there is a fatty acid tied to it, making it more hydrophobic and not ampiphathic.

Front 145

Lecture 12. The LDL is surrounded by a lipid bi or monolayer? Why?

Back 145

LDL is surrounded by a lipid monolayer since its already hydrophobic stuff inside.

Front 146

Lecture 12. What are the classic symptoms of FH (familial hypercholesteremia) in heterozygotes? Homozygotes?

Back 146

-Heterozygotes:
1) 2X number of LDL in bloodsteam.
2) Heart attacks at a middle to young age.
Homozygotes:
1) 6 to 10 times increase in LDL levels in blood.
2) Heart attacks at 6 to 10 year old.

Front 147

Lecture 12. What is wrong with FH fibroblasts in response to LDL?

Back 147

The fibroblasts cannot regulate HMG Co-A reductase activity in response to LDL/lipoprotein levels. If deplete serum used to group FH fibroblasts of lipoproteins, HMG Co-A reductase activity is not increased, and similar its activity is not decreased when LDL is added to the serum.

Front 148

Lecture 12. How many separate domains are seen in the LDL receptor? What are they? How did scientists see where relative to the membrane the C and N termini (with 7 repeats for binding) are?

Back 148

5 separate domains in the LDL receptor.
1) Ligand binding region.
2) EGF precursor homology.
3) O-linked sugars region.
4) Membrane spanning region.
5) Cytoplasmic region.
Ab staining.

Front 149

Lecture 12. Explain the cycle of how the cell uptakes cholesterol from the bloodstream.

Back 149

1) Cholesterol (in the form of LDL) binds to the LDL receptors on the membrane in a clathrin coated pit.
2) Endocytosis occurs, which transport the LDL material into a clathrin coated vesicle, which sheds its coat and binds with an endosome.
3) This endosome becomes a late ensosome and lysosome, which breaks down the LDL and the material inside. While at the late endosome stage, the low ph makes the LDL receptor let go of its cargo.
4) The LDL receptors in the LATE ENDOSOME, NOT LYSOSOME, are returned via vesicles to the PM, where at neutral ph it is free to bind to ligand again.

Front 150

Lecture 12. Scientists used what experimental technique to show the process of LDL uptake by cells?

Back 150

Pulse chase.

Front 151

Lecture 12. How exactly does the LDL receptor let go of LDL at a lower pH?

Back 151

The surface of the beta propeller domain becomes positively charged, which then binds to the ligand binding arm, but by first letting go of the LDL first.

Front 152

Lecture 12. How was the sorting signal in the LDL receptor found?

Back 152

Mutagenesis. Mutation of the cytoplasmic domain of the LDL receptor made it impossible to assemble into clathrin coated pits. This was confirmed by putting this sorting sequence into proteins normally not sorted into the clathrin pits and it was sufficient to do so. This sequences was determined to be Asparagine-Proline-X-Tyrosine (NPXY).

Front 153

Lecture 12. Many genes whose expression is controlled by cholesterol and the LDL receptor have a DNA sequence upstream called __________.

Back 153

SRE.

Front 154

Lecture 12. What are the two responses that cells have to a low intracellular levels of cholesterol?

Back 154

A transcription called SREBP binds to the SRE region upstream of the gene encording HMG-CoA reductase to increase its transcription so more cholesterol can be made. The transcription also binds to region upstream of the LDl receptor gene so more cholesterol internalization can occur.

Front 155

Lecture 12. Outline the steps how intracellular cholesterol regulates gene expression.

Back 155

SREBP, which is a transmembrane protein usually residing in the ER. It hsa two transmembrane domains and the cytosolic side of protein can bind to DNA and change transcription.
Usually SREBP is in a complex with insig-1 and SCAP, which is stabilized by cholesterol. When cholesterol levels are low, the SCAP and insig interaction weakens and SCAP and SREBP are trafficked to the golgi via copII vesicles. When the complex arrives in the golgi, S1P and S2P intermembrane proteases cleaves the SREBP from the SCAP, which releases it from the golgi membrane with a NLS on it.

Front 156

Lecture 12. Explain how statins do their job as cholesterol loweing agents.

Back 156

1) Increase SREBP mediated transcription.
2) Suppress HMG Co-A reductase levels to reduce intracellular cholesterol levels.
3)Increase expression of LDL receptor genes in all cells to reduce the levels of cholesterol in the bloodstream.

Front 157

Lecture 11. How can one tell the pH of a solution is getting lower and lower as it progresses from late endosomes to the lysosome?

Back 157

Put a litmus solution in the endosome and watch it change color.

Front 158

Lecture 11. Phagocytosis is usually a type of _________ endocytosis.

Back 158

Receptor mediated.

Front 159

Lecture 11. Outline the steps for a cell to successfully phagocytose another molecule.

Back 159

1) The Fc receptors bind to the antibody molecules, which are usually attached to the molecule.
2) Geneation of phosphotidyl inositol lipids regulates the polymerization of actin and forms the pseudopods used for phagocytosis.
3) The phagosomes fuse with the lysosomes, which degrade ingested material. Some of the digested materials are absorbed and kept in the cell, while others are exocytosed.

Front 160

Lecture 11. What are buildig components of clathrin? Can these polymerize even in the absence of vesicles?

Back 160

Three large and small polypeptides.
In vitro-yes.
In vivo-no.

Front 161

Lecture 11. What do adaptor proteins do for clathrin coated vesicles?

Back 161

1) It forms a coat between the clathrin and the membrane.
2) Trap specific transmembrane proteins as well sa soluble cargo via cargo receptors.
3) Mediate the interactions between the cargo receptor and clathrin.

Front 162

Lecture 11. What does dynamin do?

Back 162

It is a GTP binding protein that facilitates membrane fusion and pinching off of vesicles.

Front 163

Lecture 11. What are the fundamental differences between Cop I and II vs clathrin vesicles?

Back 163

1) Cop I and II vesicles have Sar1, which localizes on the Er membrane and nucleats the assembly of the coat.
2) Clathrin vesicles have dynamin to pinch off the vesicle, and Cop I and II vesicles don't need this.

Front 164

Lecture 11. What is meant by the nucleation process by Sar1?

Back 164

Nucleation of the coat proteins happens when Sar1 binds to the Er membrane in its GTP bound state and inserts its ampipathic tail.

Front 165

Lecture 11. What are caveolae and what do they do? What are they made of? What plays a major role about them?

Back 165

Caveolae are an important way to form pinocytic vesicles, which is clathrin free endocytosis.

-They do not have a coat protein.
-Formation and maintenance is primarily due to the protein caveolin, which are integral membrane proteins found in caveolae.
The lipid composition plays a major role.

Front 166

Lecture 11. Is it possible (in vitro) for clathrin bind to the LDL receptor even without cargo?

Back 166

Yes, because the LDL receptor has a binding site allowing it to bind to adaptor proteins that allow it to be recruited into a clathrin pit.

Front 167

Leture 11. Outline the process of Fe uptake by cells via endocytosis.

Back 167

1) Extracellular Fe is transported in the bloodstream attached to a carrier protein called transferrin.
2) Transferrin receptors on the cell surface mediate the delivery of transferrin into the late endosome.
3) At low pH, fe dissociates from the transferrin and is taken up into the cytosol.
4) Apotransferrin returns to the surface when its binding ability to the receptor is decreased at neutral pH-it dissociates from the receptor and can bind more Fe.
-Apotransferrin (without Fe bound) and tranferrin (with fe bound) are always bound to the receptor the entire time.

Front 168

Lecture 11. Can the iron uptake receptor be recruited into clathrin pits even without cargo (in vitro)?

Back 168

Yes, because it can bind to adaptor proteins.

Front 169

Lecture 11. In iron uptake, how does the transferrin let go of the bound Fe, while it is in the endosome still bound to the iron uptake receptor?

Back 169

Exposure to low pH allows the transferrin to convert to apotransferrin.

Front 170

Lecture 11. How is apotransferrin released from the iron uptake receptor?

Back 170

At neutral pH (outside cell), the affinity for transferrin of iron uptake receptor is high but low for apotransferrin.

Front 171

Lecture 11. How does the endosome containing the iron uptake receptor and apotransferrin localize back to the cell surface?

Back 171

Specific Rab proteins and specific v snares.

Front 172

Lecture 11. Explain how reecptor downregulation works. What greatly facilitates this process?

Back 172

An endocytosed receptor still have cytosolic domain exposed, allowing it to signal. To stop this, the endocytosed vesicle forms another vesicle containing the recptor, forming a MVB. this process is often facilitated by protein complexes called ESCRTs.

Front 173

Lecture 11. What is often the signal for receptor endocytosis?

Back 173

Reeceptor ubiquidation.

Front 174

Lecture 11. What is transcytosis? How does it work? Give an exmaple.

Back 174

A process by whih macromolecules are transferred from one extracellular compartment to another via an intracellular pathway.
-Fc receptors bind to Ab and have signals so the endocytosed materials don't go to the LATE endosome but to the recycling endosome on the other side of the cell. Exocytosis occurs when it reached other side of cell surface. Endocytosed materials can go to the EARLY endosome, which then transport materials to the recycling endosomes.

Front 175

Lecture 11. Explain how insulin causes more internalization of glucose?

Back 175

1) Increase glucose levels in the blood triggers insulin production.
2) Insulin signaling causes the delivery, to the PM of GLUT 4 molecules that have been sequestered in endosomes. These receptors allow fat and muscle cells more glucose uptake.

Front 176

Lecture 11. What is the difference between constitutive and regulated secretion?

Back 176

-Constitutive secretion is when vesicles that leave the trans golgi network migrate to the PM. Their membrane contents are incorporated into the PM and their soluble contents are released into the outside of cell. This is thought of as the default pathway for anything that goes from the golgi to the PM for exocytosis.
-Regulated secretion is for specialized secretory eclls and is when vesicles that leave the trans-golgi only fuse with the PM in response to a stimuli. Hormones or changes in membrane potential can cause an increase in intracellular calcium, which can promote fucion. (a vesicle protein can be used as the calcium level sensor).

Front 177

Lecture 11. What are secretory granules? How do they work? Are they more associatede with the constitutive or regulated secretion pathway?

Back 177

-Secretory granules are found in cell that make a lot of protein and need to export them in large, concentrated amouns. They these vesicles bud off from the golgi, they have signals that indicate that they are secretory granules (not part of the constitutive pathway). They do this by constantly having immature secretory vesicles send clathrin coated vesicles back to the golgi to retrive more cargo. When the vesicle is concentrated enough with cargo, it becomes a mature secretory vesicle, and is found in the apical regions of the PM, witing for a signal for it to fuse with PM and release contents.
-They are more associated with the regulated secretion pathway.

Front 178

Lecture 11. What is proteolytic processing? Do different cells express the same processing exzymes and cleave the same protein precursor the same way?

Back 178

Proteolytic processing is a process by which proteases are included into the secretory vesicle to cleave the proteins precursors in the vesicle to activate it.
-No, different cells may express the processing enzymes differently and cleave the same protein precursor differently.

Front 179

Lecture 11. Where can proteolytic processing occur? What are its advantages?

Back 179

Proteolytic processing can occur anywhere starting from the trans golgi all the way to when the proteins has been secreted to the extracellular domain.

Front 180

Lecture 11. What are synaptic vesicles and the advantages it conferrs upon cells such as neurons?

Back 180

Synpatic vesicles are an advantage because its allows the rapid reformation of another vesicle (via endocytosis) to be loaded with cargo to be exocytosed right after the vesicle has been used to export cargo. Entire cycle takes about 1 minute. It bypasses the need for the cell to continually sending vesicles containing cargo from the golgi, which might be a few feet away in the neuron.

Front 181

Lecture 11. In synpatic vesicles, the first vesicles containing cargo must come from where? and is secreted to the PM via a constitutive or regulated secretory pathway?

Back 181

The first vesicle containing cargo must come from the golgi and is secreted to the PM via a constitutive pathway.

Front 182

Lecture 13. Describe the in-vitro system for studying the import o proteins into mitochondria.

Back 182

1) Mitochondrial proteins with uptake import sequence made with cytoplasmic ribisomes were placed inside a test tube.
2) Mitochondria were added with ATP.
3) The mitochondrial proteins were taken into the organelles and the targeting sequence was degraded after being removed.
4) Later trypsin was added to the original test tube with un-incorporated proteins and other test tube with incorporated mitochondrial proteins. Only the test tube with unincorporated mitochondrial proteins were degraded by the trypsins.

Front 183

Lecture 13. In the in vitro assay for studying import of proteins intoi mitochondria, what was used to distinguish endogenous proteins from mitochondrial proteins that need to be imported?

Back 183

Fluorescent tags.

Front 184

Lecture 13. What is thought to be the machinery that makes ATP from a H+ gradient?

Back 184

The F1 ATPase in the inner mitchondrial membrane, which runs in reverse to drive ATP synthesis from H gradient.

Front 185

Lecture 13. What are two sources of genes that code for mitochondrial proteins? Which source codes for more mitochondrial proteins?

Back 185

1) Genes can reside in the nuclear DNA, which are translated along with a N terminus mitochondrial transport sequence. This source codes for mre mitochondrial proteins.
2) Genes are on the mitochondrial plasmids.

Front 186

Lecture 13. The mitochondrial mport signal has ____-____ (a number) amino acid. A portion of it can form an ________ with positively charged AAs on one side and ________ onthe other side. This helix binds with its ______ surface to a ________ in the receptor on the mitochondria.

Back 186

The mitochondrial import signal has 20-80 AAs. A portion of it can form a alpha amphipathic helix with positively charged AAs on one side and hydrophobic AAs on the other side. The helix binds with its hydrophoib side to the hydrophobic groove on the receptor on the mitochondria.

Front 187

Lecture 13. Describe the in-vitro system for studying the import o proteins into mitochondria.

Back 187

1) Mitochondrial proteins with uptake import sequence made with cytoplasmic ribisomes were placed inside a test tube.
2) Mitochondria were added with ATP.
3) The mitochondrial proteins were taken into the organelles and the targeting sequence was degraded after being removed.
4) Later trypsin was added to the original test tube with un-incorporated proteins and other test tube with incorporated mitochondrial proteins. Only the test tube with unincorporated mitochondrial proteins were degraded by the trypsins.

Front 188

Lecture 13. In the in vitro assay for studying import of proteins intoi mitochondria, what was used to distinguish endogenous proteins from mitochondrial proteins that need to be imported?

Back 188

Fluorescent tags.

Front 189

Lecture 13. What is thought to be the machinery that makes ATP from a H+ gradient?

Back 189

The F1 ATPase in the inner mitchondrial membrane, which runs in reverse to drive ATP synthesis from H gradient.

Front 190

Lecture 13. What are two sources of genes that code for mitochondrial proteins? Which source codes for more mitochondrial proteins?

Back 190

1) Genes can reside in the nuclear DNA, which are translated along with a N terminus mitochondrial transport sequence. This source codes for mre mitochondrial proteins.
2) Genes are on the mitochondrial plasmids.

Front 191

Lecture 13. The mitochondrial mport signal has ____-____ (a number) amino acid. A portion of it can form an ________ with positively charged AAs on one side and ________ onthe other side. This helix binds with its ______ surface to a ________ in the receptor on the mitochondria.

Back 191

The mitochondrial import signal has 20-80 AAs. A portion of it can form a alpha amphipathic helix with positively charged AAs on one side and hydrophobic AAs on the other side. The helix binds with its hydrophoib side to the hydrophobic groove on the receptor on the mitochondria.

Front 192

Lecture 13. Are the 20 to 80 amino aicds on the N terminus of the mitochondrial import sequence hydrophobic or hydrophilic?

Back 192

Hydrophobic.

Front 193

Lecture 13. How can one prove that the N terminus mitochondrial import sequences are necessary?

Back 193

1) Can make GFP-N terminus, the protein goes into the mitochondrial.
2) If you take the mitochondrial protein and make a deletion at the N terminus sequence you see that the protein does not end up in the mitochondria.

Front 194

Lecture 13. Mitochondrial proteins have to traverse _______ (a number) membranes, whereas nuclear proteins only have to traverse ______. However, mitochondrial proteins are usually imported into the mitochondria at ________, where the outer and inner mitochondrial membranes are apposed.

Back 194

Mitochondrial proteins have to traver 2 membrane in the transport into the mitochondria whereas nuclear proteins only need to traverse the nuclear pore, which is one obstable that covers both nuclear mambranes. Contact points.

Front 195

Lecture 13. Protein import into the mitochondrial matrix requires proteins in the ________ and _______ that utilize _____ for energy.

Back 195

Mitochondria and cytosol. ATP .

Front 196

Leture 13. Mitochondrial proteins pass through the pores at the contact point in a folded or unfolded state? What proteins make this possible, what do they require to function?

Back 196

Unfolded. This requires ATP. Chaperone proteins in the motichondria and cytosol. ATP.

Front 197

Lecture 13. How is mitochondrial transport different from the transport of proteins into the nucleus or ER?

Back 197

In protein transport into the ER, the proteins are threaded in an unfolded state POST-TRANSLATIONALLY. In nuclear import/export, proteins are imported after translation but are in a folded state.

Front 198

Lecture 13. How do people know that proteins go into the ER during, NOT AFTER translation?

Back 198

Making microsomes and seeing that proteins already translated and folded do not go in.

Front 199

Lecture 13. How exactly do chaperone-like proteins keep mitochondrial proteins about to be transported in an unfolded state?

Back 199

They grab onto different parts of the protein and make it energetically unfavorable for proteins to fold by inhibiting the interactions between the interacting domains on the protein.

Front 200

Lecture 13. When does the mitochondrial protein resume normal conformation/folding?

Back 200

After 1) fully entering the mitchondria.
2) N terminus transport sequence is cleaved by mitochondrial proteases in the matrix.

Front 201

Lecture 13. How do proteins in the receptor at the contact point of mitochondria thread the protein through both membrane, what do they need?

Back 201

They need ATP and afterwards, they ratchet the protein through both membranes concurrently. They use ATP to do their work.

Front 202

Lecture 13. What is the main difference between when the signal sequence is cleaved in mitochondrial transport and ER transport of proteins?

Back 202

In ER transport, the signal peptidase cleaves the signal sequence WHILE the protein is being threaded through. In mitochondrial protein transport, the signal sequence is cleaved AFTER transport into the matrix.

Front 203

Lecture 13. What was the experiment that showed that proteins needed to be unfolded to be transported into the mitochondria?

Back 203

People added methotrexate, which stabilizes the folded state of the protein and saw that the protein cannot enter the mitochondria.

-Researchers used DFTR, which was tagged to an Ab for contrast and viewed under EM. They saw that only the naturally unfolded regions of the protein was threaded through the contact point, the rest was outside, clogging up the pore.

Front 204

Lecture 13. Do peroxisomes have DNA? RNA? ribosomes? How do they acquire proteins?

Back 204

No, no, no. They acquire proteins by selective import from the cytosol.

Front 205

Lecture 13. What happens in the peroxisomes usually? How are they generated?

Back 205

-ou can see peroxisomes by tagged it to GFPs.
-Peroxisomes are generated by having vesicles budding off from the ER, which already have proteins on its membrane that catalyze protein import. These vesicles mature into peroxisomes by incorporating specific peroxisomal proteins and lipids from the cytosol.

-Daughter peroxisomes can be generated from this peroxisome by fission. The same process can be seen in mitochondria.

Front 206

Lecture 13. The import of proteins into peroxisomes require (necessary? suffient? both?) what amino acid motif? Is this targeting sequence on the N or C terminus of the protein?

Back 206

The AA motif S-K-L is both necessary and sufficient for peroxisomal protein import. The targeting sequence is on the C terminus of the protein.

Front 207

Lecture 13. Peroxisomal proteins are imported in a folded or unfolded state? Are signal sequences cleaved after import?

Back 207

Folded. No.

Front 208

Lecture 13. Proteins that are to be transported into the mitochondrial matrix are unfolded starting where?

Back 208

The proteins are unfolded starting in the cytosol.

Front 209

Lecture 13. How do perixisomes interact with the peroxisomal receptors to be imported?

Back 209

The peroxisomal proteins bind to the Pex5 receptor which is in the cytosol, and this receptor interacts with thePex14 receptor on the membrane. This complex interacts with the Par12 to go into the perixisomal lumen and release cargo, after the Pax5 receptor is able to exit the peroxisome via the Pex2 receptor.

Front 210

Lecture 5. What is one example of one transporter relying on other transporters to work?

Back 210

1) The passive flow of K+ out of the cell using the gradient set up by the Na+/K+ ATPase (needs ATP). This ultimately sets up a voltage difference between the two sides of the membrane (passive diffusion).
2) The Na+ gradient set up by the Na+/K+ ATPase is used by the Na+/lysine symporter to import Na+ into the cell against a concentration gradient. (secondary active transport -relies on ATP spent on primary active transport).

Front 211

Lecture 5. Glucose uniporters act as enzymes because __________. Why do liver cells experience the greatest change in glucose import compared to other types of cells when the glucose concentration outside increases?

Back 211

They exhibit similar kinetics. Glucose uniporters in the hepatocytes have the highest Vm (1/2 max transport rate).

Front 212

Lecture 7. In a reconstitution experiment, researchers found out that adding what things were sufficient. Of course, this was done with microsomes present, what would you need if you only had liposomes?

Back 212

mRNA, reticulocyte lysate, and SRP.

Sec61 channels.

Front 213

Lecture 7. What are evidence that a SRP receptor existed?

Back 213

1) If you have SRP and ribosome complexes in a test tube and you add SRP receptor, you can displace the SRP from the peptide/ribosome complex.

-Both SRP receptor subunits and SRP can bind GTP!!

Front 214

A protein's topology is determined once it is placed in the ______.

Back 214

ER.
-Only parts of the protein inside the lumen of the ER can be modified by its enzymes.

Front 215

Lecture 8. What do the disulfide bonds on proteins in the extracellular domain do?

Back 215

It stabilizes the proteins (via intra-chain and inter-chain disulfide bonds). Initial order of these bonds may differ from the final order due to ER enzymes constantly breaking and re-making these bonds.

Front 216

Lecture 8. Can some of the processes that happen in the ER happen elsewhere further down the secretory pathway?

Back 216

Yes, such as the glycosylation and proteolytic cleavage.

Front 217

Lecture 9. Do the coats on vesicles stay on forever?

-When is the GTP on Sar 1 hydrolyzed?

-What is the conformational change that occurs to Sar1 when the GTP binds?

Back 217

No. After the vesicle buds off. Sar1 extends a little helix that allows it to anchor in the PM.

Front 218

Lecture 9. Sec24 has binding sites for transmembrane or soluble proteins or both? How does the soluble protein cargo bind?

Back 218

Both. Soluble protein cargo must bind to the cargo receptor first, which can bind to the sec24 coat protein.

Front 219

Lecture 9. The snares v and t snares are held together via covalent or non-covalent interactions? What initiates the membrane fusion event?

Back 219

Non-covalent. The wrapping of v and t snares initiates membrane fusion.

Front 220

Lecture 9. Different enzymes involved in oligo-saccharide processing are localized different parts of the Golgi, how did researchers know this? Which enzyme located in which compartment of the Golgi generates an oligosaccharide that is insensitive to endoH? How can scientists confirm that protein has crossed the medial golgi?

Back 220

Pulse-chase. Mannosidase II at the medial golgi. The times that it takes for them to see a mobility shift in gel.

Front 221

Lecture 10. Where does the M-6-P bind to in the ____ Golgi? and fuses with what compartment? The hydrolases dissociates from the M-6-P receptor in the ____ pH and the ______ is removed by the acid phosphatase. What is the point of this? The unoccupied receptor is then _______.

Back 221

M-6-P receptor on the trans golgi forming a clathrin coated vesicle that fuses with an endosome or PM.

Acidic. Phosphate, which is removed so the hydrolases cannot re-bind to mannose.

The unoccupied receptor is returned to the golgi by incorporating into a retromer vesicle or it can go to the PM.

Front 222

Lecture 10. What is the second way of getting lysosomal enzymes to lysosomes?

Back 222

There are M-6-P receptors on the PM surface for any enzymes that escape. These move into the cell and incorporates into clathrin coated vesicles that fuse with the endosomes.

Front 223

Lecture 10. What is I cell disease?

Back 223

Patients are found with large inclusions in their cells, because they are never destroyed by the lysosomes.

There is a defect of transporting lysosomal enzymes into the lysosome. There is a defect is thelysosomal protein signal. The GlucNac Phosphotransferase has a problem. So they dont even get a M-6-P on protein. They are instead secreted and never come back.

Front 224

Lecture 10. What is Caigher's disease?

Back 224

Patients don't have glycocerobrosidase, a hydrolase. To treat it, you synthesize it and tag it with M-6-P.

Front 225

Lecture 12. Which part of the LDL particle allows it to bind to the LDL receptor?

Back 225

ApoB.

Front 226

Lecture 2. What destinations within or outside the cells is only possible to proteins cargoes after going through the Golgi?

Can the cargo go straight from golgi to late endosomes? Lysosomes?

Back 226

1) Secretory vesicles.
2) Late/early endosomes.
3) Cell exterior.

-Yes. No.