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MBIOS 501 - Cell Biology > W4: Endoplasmic Reticulum > Flashcards

W4: Endoplasmic Reticulum Flashcards

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1
Q

What is the difference in structure between the RER and SER?

A

RER has a membranous sac and is rough looking b/c of ribosomes

SER tubular structure and found in interconnected tubules.

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2
Q

If you centrifuge RER and SER which one will be found at the bottom of tube?

A

RER is slightly denser/heavier b/c or ribosomes.

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3
Q

What are the functions of the SER?

A

Site of lipid, cholesterol, carbohydrates, biosynthesis

site of Ca2+ channels” Ca2+ level higher in ER (used in cell signaling and muscle contraction and can be released in cytosol in response to particular signal).

SER is a detoxification center for xenobiotic compounds (foreign substances, drugs)

A channel between ER and Golgi sometimes called transitional ER

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4
Q

How does P450 (monooxygenase enzymes) help SER detoxify?

A

P450 hydroxylates hydrophobic toxins and then they are flushed out by kidney.

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5
Q

What are the major functions of the Rough ER?

A
  • site of protein synthesis and translocation (con translational translocation)
  • proteins in the endosomal pathway are made and translocated here
  • ER lumen is a site of post-translational protein modification, such as glycosylation, disulfide bonds, etc.
  • ER lumen is a site of surveillance for misfolded proteins
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6
Q

How is translocation of a protein into the ER initiated?

A

The translocation is most commonly coupled to translation of the mRNA into a protein (protein synthesis). N-terminal signaling peptide is synthesized and targets the protein to the ER.

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7
Q

How does the n-terminal signal and signaling recognition particle help translocate a protein?

A

N-terminal signal peptide will be identified by signaling recognition particle (SRP) and will attach to the leader peptide + ribosome. This attachment will halt protein translation temporarily.

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8
Q

What happens after SRP + Ribosome + N-terminal leader peptide are binded?

A

SRP Receptor (which is in the ER membrane) will bind with SRP which is attached to Ribosome + N-terminal leader. The ribosome and protein are then transferred to the protein translocator complex (which is also embedded in the ER membrane). SRP and SRP receptor are released.

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9
Q

Once the protein and ribosome are attached to the protein translocator complex (aka transmembrane transporter complex), how does the protein move across the ER membrane?

A

Protein synthesis will be resumed (translation) and the energy used to move the protein out of the ribosome as it is synthesized is used directly to move the protein across sec61 core complex. (translocation)

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10
Q

What are the two exit pathways of Sec61 protein transporter?

A
  • Central Pore that goes through the membrane
  • lateral seam between the two of the major subunits that will allow a peptide sequence to interact directly wit the lipid bilayer during transport
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11
Q

What is the structure of Sec61 and how does it contribute to exit ways?

A

Made up of tetramer of two large and two small protein subunits. Rearrangement of the subunits can open pore and lateral seam

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12
Q

When does sec61 interact with a synthesized protein?

A

A the N-terminal signaling peptide.

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13
Q

N-terminal peptide is recognized by what two proteins?

A

SRP-helps transport N-terminal peptide and ribosome to the ER membrane

Sec61-helps translocate (transport) synthesized protein through ER membrane

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14
Q

Describe how sec 61 and synthesized protein interact. How is most of the protein actively fed through the ER? What ends up happening the the n-terminal signal?

A

association of sec61 and n-terminal part of synthesized protein acts as a start signal and opens the central pore. Most of synthesized protein is fed through the central pore into ER lumen.

N-terminal signaling peptide will shift during this process and ends up in the lateral seam in the sec61 wall.

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15
Q

Does sec61 require energy to translocate protein?

A

No, since ribosome is attached w/n-terminal signal peptide protein sytnehsis will proceed. The energy used to to move protein our of ribosome as it is synthesized will be used directly by sec 61. Sec61 does not require energy to function itself.

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16
Q

How do soluble proteins transport into the lumen of the ER?

A
  • leader signal recognized by sec61 and binds the imported signal peptide
  • bulk of the protein is transported through the central pore into lumen of ER.
  • signal peptidase cuts off the signal peptide which the leaves sec 61 through lateral seam.
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17
Q

When else is the transport mechanism used to transport soluble proteins used?

A

involved in all secreted pathways as well as the synthesis proteins that are residents of the endosomal membrane system

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18
Q

How are GPI linked proteins transported into the ER? How is it different than transporting soluble proteins?

A

Uses similar transport mechanism as when transporting soluble proteins. The main difference is the GPI linked protein has a C-terminal signal sequence used to identify it, where as soluble proteins have an N-terminal signal.

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19
Q

What is the transport mechanism for transmembrane proteins or GPI-ANCHORED (not linked) proteins?

A
  • N-terminal “start” transfer sequence is needed.
  • A “stop” transfer signal is needed and is located somewhere within the protein.
  • N-terminal “start” associates with sec61 and protein pass through central pore until it reaches the “stop” signal sequence & translocation stops
  • At this point N terminal start sequence is cut of by signal peptidase.
  • Region containing stop sequence is not cut off but leaves sec61 via lateral seam into the membrane. The result is a single pass transmembrane protein
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20
Q

What is the positive inside rule?

A

positively charged protein domain next to the start sequence will always remain in the cytoplasm.

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21
Q

What happens if positive charged protein region is synthesized after the “start” sequence?

A

The amino- or N-terminal end of the protein will be passed into the ER of the lumen (which is spatially equivalent to the outside of the cell.

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22
Q

What happens if the positive charged protein region is synthesized before the “start” sequence?

A

the protein will be in its opposite orientation. The amino-terminal end will be in the cytoplasm. Cytosolic face of the cells outer membrane is negatively charged (opposite attracts)

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23
Q

In the positive inside rule, inside equals what?

A

inside = cytosolic side of the membrane

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24
Q

What happens if a protein has more than one internal start sequence? What if there are multiple signal sequence?

A

Proteins that have more than one signal sequence can produce multi-pass transmembrane proteins.

25
Q

Are all proteins transported during their synthesis to the ER?

A

No, some are transported into the ER after being translated by a free ribosome in the cytoplasm.

26
Q

What is the process of transporting and already synthesized protein into the ER (aka post-translational translocation into the ER)?

A

-Involves presence of “start” signal at the N-terminal end of the protein.

New player: transmembrane complex compromised of four different sec proteins binds to the protein during the signal recognition phase.

The luminal end of this complex recruits an ATPase protein called BiP to the complex, which uses the energy of ATP to pull the target protein into the ER.

27
Q

What are some modifications of proteins that happen inside the ER?

A
  • glycosylation
  • formation of GPI-linkages
  • monitoring of proper folding state
28
Q

What is glycosylation and why is this done to proteins inside the ER?

A
  • Glycosylation is the process of adding carbohydrates (CHO)
  • CHO assists in proper folding and transport of proteins
  • CHO protects proteins from digestion of protease
  • CHO increases the solubility of proteins
  • CHO can be a recognition site (cell-cell recognition and cell signaling)
29
Q

During glycosylation, the oligosaccharide chain is linked to which atom in the protein?

A

Nitrogen, giving it the name N-linked glycosylation

30
Q

Which amino acid is involved in glycosylation ?

A

Asparagine

31
Q

Describes the steps in glycosylation

A

1) transfer of a precursor oligosaccharide with 14 sugars to the protein to (connects to asparagine) from dolichol

The first two sugar residues that are closes to the asparagine link are N-acetyl glucosamine, followed by 9 mannose sugars, and 3 glucose sugars at the end.

32
Q

How is the the 14 sugar precursor formed?

A

Foundation of the precursor is dolicol (unique lipid that spans the whole lipid bilayer several times).

1) addition of two high energy phosphate molecules at the first N-acetyl glucosamine residues (happens on the cytoplasmic face of the ER membrane)
2) Five mannose residues are added next and the the whole complex is flipped into the ER lumen
3) once in the ER lumen addition of sugar residues is completed by the addition of four more mannose residues and three glucose residues

33
Q

How does adding oligosaccharides to proteins entering the ER help act as a checkpoint for the exit of the protein from the ER?

A
  • Three different proteins w/in the ER are involved in retaining unfolded proteins w/in lumen until folded properly.
  • To start this process, the oligosaccharide bound to a protein is trimmed until only one terminal glucose is present.
  • Calnexin and Calreticulin bind to proteins having single terminal glucose on their associated oligosaccharide chains.
  • while bounds, a protein has time to fold properly. Whether or not it does, the protein is eventually released from Calnexin (by enzyme glucosidase) and this cuts off the last glucose.
  • If protein is folded properly, the protein escapes the cycle and leaves the ER. If its not, it will be recognized by a third protein (glucosyl transferase) and this adds back the terminal glucose and the cycle repeats. T

This mechanism ensures that unfolded proteins don’t leave the ER very often.

34
Q

What are the three player proteins that help in protein folding?

A

Calnexin and Calreticulum which bind to proteins with single terminal glucose

and Glucosyl transferase which adds back the glucose if the protein is unfolded at the end of the folding cycle

35
Q

How does the ER deal with proteins that never fold?

A

Export them back out of the ER and target them to a protein degradation pathway. This pathway is called ERAD

36
Q

What is ERAD: ER-Associated degradation?

A
  • misfolded protein exits the ER (retranslocation). It is pulled out of the ER by chaperone and protein transporter( AAA (ATPase)))
  • once in the cytoplasm the protein is deglysosylated
  • ubiquinatin then attaches to the protein
  • Proteasome recognize ubiquinatin and degradate protein
37
Q

what happens when there are too many unfolded proteins in the ER?

A

cells use three different mechanisms to change gene expression patterns:

1) IRE1 (mRNA splicing)
2) PERK (modulated ribosome function)
3) ATF6 (regulates gene expression with transcription factor)

38
Q

What is the similarity between 1REI, PERK, ATF6 in gene expression?

A

They all follow the first step of gene expression: binding of unfolded proteins to the part of the signaling proteins which are inside of the ER lumen. (only step inside of the ER all others are outside in the cytoplasm)

They also have the same final result: activated transcription factors will enter the nucleus where they increase expression of things like chaperones that end up back in the ER to help proteins fold properly.

The middle part is where they all differ.

39
Q

What does IRE1 do?

A

IRE1 is autophosphorylated and forms oligomers when it binds to unfolded proteins.

oligomers process RNA that encodes a transcription factor, which will enhance the expression of chaperones

40
Q

What does PERK do?

A

PERK is a kinase. when it binds to unfolded proteins it phosphorylates and therefore inactivates proteins that are necessary for ribosome to make more proteins.

This decreases the amount of protein that enters the ER.

When ribosomes are inhibited, they still make some protein but prefer to make transcription factors that enhance chaperone expression.

41
Q

What does ATF6 do?

A

ATF6 is a transcription factor itself.

It is normally inactive and held in the ER.

When it binds to unfolded proteins on the side in side the ER, it is transported out of the ER to the Golgi apparatus and there the cytoplasmic domain of the protein is cleaved from the rest.

The cleaved portion is an active transport factor.

42
Q

SRP stands for? Where is this used?

A

Signal Recognition particle and it is used in the co-translational protein translocation into the RER.

43
Q

N-terminal signaling peptide is used for what?

A

translocation proteins into the ER

44
Q

How are proteins that leave the ER modified?

A

They are glycosylated (adding carbohydrates to protein CHO)

45
Q

Why is glycosylation that happens in the ER called N-linked glycosylation?

A

because
the oligosaccharide chain is linked to the nitrogen atoms in the protein.

This is by far the most common linkage used to create glycoproteins in cells and the amino acid involved is always asparagine.

46
Q

What is the first step in N-linked glycosylation?

A

The initial step in glycosylation is the transfer of a
precursor oligosaccharide with 14 sugars to the protein from a specialized membrane lipid called
dolichol

47
Q

What is the role of dolichol in n-linked glycosylation?

A

The oligosaccharide with 14 sugars is transferred to the asparagine part of the protein by dolichol

48
Q

What enzyme helps catalyze the transfer of the 14sugar oligosaccharider from dolichol to the asparagine part protein being glycosylated?

A

oligosaccharyl transferase

49
Q

What is GlcNAc?

A

N-acetylglucosamine

50
Q

What is the role of dolicol? (dolicol without the h, remember there is also a dolichol but there’s a difference)

A

helps form the 14 sugar precursor.

Dolicol (poly-isoprenoids) phosphate is a hydrophobic lipid with 75-95 carbon atomembedded in the ER membrane.

51
Q

When creating 14 sugar precursor (oligosaccharide) which two sugar residues are added on the cytoplasmic side and how many of each?

Which are added in the ER lumen?

A

2 high energy phosphates must be added first before the first n-acetyl glucosamine

Cytoplasmic side: (2) N-acetyl glucosamine residues and (5) Mannose residues

complex is flipped

ER lumen: (4) mannose residues and (3) glucose residues

52
Q

How does Glucosyl tranferase assist in protein folding in the ER?

A

adds glucose to a misfolded protein so that it can be recognized by calnexin or calreticulin

53
Q

Why is it important to add oligosaccharides to proteins coming into the ER?

A

helps ensure proteins are folded properly before leaving the ER

54
Q

Does a properly folded protein exposes their hydrophobic domain or does it hide it interiorly?

A

Properly folded means hydrophobic domain is in the interior.

55
Q

What happens to proteins that expose their hydrophobic domain in the ER lumen?

A

Proteins that expose hydrophobic domains are not properly folded and can be recognized by other proteins known as chaperones that assist folding of unfolded proteins.

56
Q

How do calnexin/calreticulin assist in protein folding in the ER lumen?

A

they bind to proteins that have one single terminal glucose molecule and hold the protein, which gives the protein time to fold. (just b/c it holds the protein does not mean the protein will fold).

57
Q

How is the protein released from calnexin/calreticulin? What can the protein do after being released?

A

cuts of the last glucose of the protein which separates it from calnexin/calreticulin. The protein can then leave the ER, if it folded properly. If it didn’t fold then glucosyl transferase will attach another terminal glucose to the protein so that calnexin/calreticulin can bind to the protein.

58
Q

What happens when there are too many unfolded proteins in the ER? AKA what is the unfolded protein response.

A

Unfolded Protein Response
 IRE1: inositol requiring enzyme. mRNA splicing
 PERK: PKR-like ER kinase. Modulated ribosome function.
 ATF6: activating transcription factor. Regulates gene expression (transcription factor)

59
Q

IRE1, PERK, and ATF6 do what?

A

activated transcription factors will enter
the nucleus where they increase expression of things like chaperones that end up back in the ER
to help proteins fold properly

MBIOS 501 - Cell Biology (14 decks)

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