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Protein secretion Flashcards

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1
Q

4 Functions of the endoplasmic reticulum

A
  1. Protein production- translation, folding, and some protein modifications. It produces all transmembrane proteins
  2. Lipid production- produces most of the lipids for the cell and its organelles
  3. Serves as a store of intracellular calcium that is involved in cell signaling responses
  4. Proteins are delivered to the ER lumen before they are excreted from the cell
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2
Q

Endoplasmic reticulum

A

A netlike labyrinth of branching tubules and flattened sacs that extends throughout the cytosol. The tubules and sacs are interconnected, and their membrane is continuous with the outer nuclear membrane. The different regions of the ER are specialized (rough and smooth ER) to meet different functional demands of the cell. The smooth and rough ER form one continuous network

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3
Q

ER lumen

A

The single internal space enclosed by the ER and nucleus membranes

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4
Q

Co-translational process

A

A process that occurs as the polypeptide chain is being formed- cells import most proteins into the ER before complete synthesis of the polypeptide chain, which is a co-translational process. During co-translational transport, the ribosome that is synthesizing the protein is attached directly to the ER membrane. This allows one end of the polypeptide chain to be translocated into the ER while the rest of the chain is still being synthesized. Co-translational import of proteins is essentially what causes a rough ER to appear. These membrane bound ribosomes are part of the rough ER

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5
Q

Post-translational process

A

The import of proteins into the mitochondria, chloroplasts, nuclei, and peroxisomes is post-translational. When production of proteins occurs post-translationally, the proteins are made by a free ribosome

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6
Q

Rough endoplasmic reticulum

A

Membrane bound ribosomes that coat the surface of the ER. This plays a role in protein synthesis, as the ribosome synthesizing proteins is attached directly to the ER membrane. The protein is translocated into the ER co-translationally.

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7
Q

Smooth endoplasmic reticulum

A

Region of the ER that does not contain membrane bound ribosomes. It is not as abundant as the rough ER. However, the smooth ER is prominent in cells that specialize in lipid metabolism, like cells that use cholesterol to synthesize steroid hormones. In this case, the specialized cells contain more smooth ER, as the smooth ER accommodates the enzymes that make cholesterol and modify it to form hormones. It is the site of production of lipoprotein particles, and it contains enzymes that catalyze a series of reactions to detoxify lipid soluble drugs and harmful compounds produced by metabolism.

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8
Q

Transitional ER

A

Areas of the smooth ER from which transport vesicles carrying newly synthesized proteins and lipids bud off for transport to the Golgi apparatus

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9
Q

Functions of the smooth ER (2)

A
  1. Membrane lipid synthesis
  2. Vesicle budding and release, carrying newly synthesized proteins and lipids, which are sent to the Golgi apparatus
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10
Q

Lipoprotein particles

A

Particles that carry lipids via the bloodstream to other parts of the body. The enzymes that synthesize the lipid components of these particles are located in the membrane of the smooth ER

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11
Q

Microsomes

A

Small, closed vesicles (100-200 nm diameter). The ER breaks into fragments when cells or tissues are disrupted by homogenization, and these fragments reseal to form microsomes. Microsomes are still functional, and they represent small versions of the ER. They are still capable of protein translocation, protein glycosylation, calcium uptake and release, and lipid synthesis. Microsomes are used to study the ER. The interior of the microsome is also considered biochemically equivalent to the lumen of the ER

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12
Q

Rough microsomes

A

Microsomes derived from the rough ER. These microsomes are also studded with ribosomes. The ribosomes attached to rough microsomes make them denser than smooth microsomes, so equilibrium centrifugation can be used to separate rough and smooth microsomes

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13
Q

Smooth microsomes

A

Vesicles that are similar in size to rough microsomes, but that lack attached ribosomes. They are derived partially from the smooth ER, but partially from vesiculated fragments of the plasma membrane, Golgi apparatus, endosomes, and mitochondria. Therefore, it’s difficult to separate smooth microsomes that are derived from different organelles, although rough microsomes are clearly derived from the rough ER

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14
Q

Density gradient centrifugation

A

Creates a discontinuous gradient of densities or viscosities. The most dense or most viscous components of the cell or tissue will end up at the bottom of the tube, while the least dense will end up at the top. When centrifugation is used to separate smooth and rough microsomes, rough microsomes are found at the bottom since they have a high density. Smooth microsomes are less dense and are found at the top

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15
Q

Free ribosomes

A

Present in the cytoplasm. They synthesize soluble, cytoplasmic, and nuclear proteins. The proteins probably won’t leave the cell or be part of a membrane- they will be completely soluble

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16
Q

Membrane bound ribosomes

A

Located on the rough ER, on the cytosolic side of the ER membrane. Synthesize proteins destined for secretion out of the cell, to go to the ER, to be part of lysosomal compartments, or to be part of organelle or cell membranes

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17
Q

What determines the function of membrane bound and free ribosomes?

A

The two types of ribosomes are functionally the same. The location of the ribosome (cytoplasm or ER membrane) dictates the type of protein that is made. There is a common pool of ribosomes that switch between each location

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18
Q

How are proteins imported into the ER lumen?

A

They can be imported post-translationally or co-translationally. When proteins are imported co-translationally, the ribosome comes to the ER membrane, causing the rough ER to appear. When proteins are imported post-translationally, the proteins are produced by free ribosomes in the cytosol and transported to the ER using chaperones. Proteins that are transported to the ER lumen are destined to stay there, to be secreted out of the cell, to be transported to the Golgi or lysosomes, or to be stuck in membranes

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19
Q

4 fates of proteins transported to the ER

A
  1. Stay in the ER
  2. Secretion out of the cell
  3. Transport to the Golgi or lysosomes
  4. Become stuck in the cell membrane
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20
Q

2 types of proteins captured from the cytosol by the ER

A
  1. Transmembrane proteins- proteins that are partly translocated across the ER membrane and become embedded in it- these proteins may also end up in the plasma membrane or in the membrane of another organelle
  2. Water-soluble proteins- proteins that are fully translocated across the ER membrane and are released into the ER lumen
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21
Q

Transmembrane proteins

A

Proteins that are partly translocated across the ER membrane and become embedded in it- these proteins may also end up in the plasma membrane or in the membrane of another organelle

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22
Q

Where are water soluble proteins destined to end up?

A

They are destined either for secretion or to stay in the lumen of the ER or of another organelle

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23
Q

ER signal sequence

A

Whether proteins go to the ER or stay in the cytoplasm has to do with the protein itself. All proteins, regardless of if they are fully or partially made, contain an ER signal sequence (also called a leader peptide). It is an amino acid sequence located at the extreme end terminus of a newly translated protein. It is usually made of around 8 nonpolar amino acids, although there is no consensus as to which amino acids they are. In a co-translational situation, the signal sequence is also important in directing the ribosome to the ER membrane

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24
Q

Signal peptidase

A

Membrane-associated protease in the ER membrane that has its active site in the ER lumen. Once a newly translated protein arrives in the ER lumen, the signal peptidase cleaves its ER signal sequence. Therefore, the signal sequence only directs the protein to the ER, it is not part of the final protein

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25
Q

Protein translocation into the ER lumen

A

When a signal sequence is produced in the ribosome, it directs the entire ribosome to the ER membrane and to secretion machinery through which the protein can be secreted into the ER lumen. Translocators form a hole in the ER membrane that the protein is directed through. The signal peptidase enzyme is bound to the ER membrane with its active site in the lumen. This enzyme helps to cut off the signal sequence as the protein enters the lumen. Therefore, a mature protein is released into the lumen as soon as it is synthesized

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26
Q

3 regions of the signal sequence/leader peptide

A
  1. Basic region
  2. H domain (core)
  3. C-domain
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27
Q

Basic region of the leader peptide

A

Composed of 1-3 positively charged amino acids at the N-terminus. It attaches to the cytoplasmic side of the ER membrane

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28
Q

N-terminus

A

The first part of the protein that exits the ribosome during protein biosynthesis

29
Q

H-domain (core) of the leader peptide

A

Composed of 10-15 hydrophobic amino acids. This portion inserts into the ER membrane

30
Q

C-domain of the leader peptide

A

Consists of 3-7 uncharged amino acids at the C-terminus end. It contains a recognition site for the signal peptidase. This is where signal peptidase will cut the leader peptide from the protein as it enters the ER lumen

31
Q

C-terminus

A

C-terminus is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). It is the opposite end from the N-terminus

32
Q

Translocase

A

A pore (aqueous channel) in the ER membrane through which an unfolded protein travels.

33
Q

Sec61

A

The version of the translocase found in mammalian cells. It is the core of the translocator which creates a pore at the ER membrane. It is built from 3 subunits which are highly conserved from bacteria to eukaryotic cells, and has a central pore. The plug component closes the pore when no proteins are being secreted. There is also a lateral seam between the 2 adjacent subunits which can open

34
Q

SecYEG

A

The version of the translocase found in prokaryotes, which acts at the cell membrane as prokaryotes do not have an ER

35
Q

Sections of Sec61 and SecYEG

A

Both Sec61 and SecYEG contain these pieces, although the names are the prokaryotic designations
1. SecYE- a protein conducting channel. This is the main component of the Sec translocon
2. SecG- stimulates transport through the YE component. Mechanism is not clear, but it may serve as a plug in the pore when there are no proteins being secreted

36
Q

Seam of the Sec 61 complex

A

There is a lateral seam in the alpha subunit of the Sec61 complex. The seam can open to allow lateral diffusion of the cleaved signal sequence into the ER membrane. Once the signal sequence has been cleaved, it can move into the hydrophobic interior of the ER membrane. Most of the signal sequence is hydrophobic, so it can easily partition into the membrane. It laterally partitions at the membrane location because there are enzymes in the ER membrane to degrade it

37
Q

2 components that guide the ER signal sequence to the ER membrane

A
  1. Signal recognition particle (SRP)
  2. SRP receptor
    These pieces of machinery are used during co-translational secretion
38
Q

Signal-recognition particle (SRP)

A

Cycles between the ER membrane and the cytosol during co-translational secretion. It binds to the signal sequence of a protein that needs to go to the ER, and it also binds to the ribosome that is producing that protein. Therefore, the SRP ferries the protein and the ribosome to the ER membrane. The SRP tells the ribosomes to go to the membrane, creating rough ER

39
Q

SRP receptor

A

A protein located in the ER membrane and exposed on the cytosolic face of the membrane. The SRP binds here along with the bound protein and ribosome. The receptor is in close proximity to the sec61 translocase

40
Q

SRP structure

A

It is a 6 subunit protein (quaternary structure) that also contains an RNA molecule, which is why it is called a particle instead of a protein. SRP contains a binding site for the signal sequence, which is a hydrophobic pocket lined with methionine. This is because methionine is very flexible, with a small side chain, which can accommodate many different types of signal sequences. It also contains another binding site called the translational pause domain

41
Q

Translational pause domain

A

A binding site on the SRP. It binds to the ribosome and stops translation by plugging the interface between the large and the small subunits of the ribosome, which prevents elongation factors from entering. If elongation factors can’t enter, then translation is halted

42
Q

SRP binding sites

A

One of the binding sites binds to the signal sequence on the new protein. The other (translational pause domain) binds to the interface between the large and small subunits of the ribosome and stops translation

43
Q

Mechanism of co-translational protein secretion

A
  1. SRP binds to the signal sequence and well as to the interface between the large and small ribosomal subunits
  2. Translation pauses, as long as the SRP is bound
  3. SRP binds to the SRP receptor in the ER membrane
  4. The receptor is in close proximity to the Sec61 pore. It is close enough that the signal sequence can take over and direct the translating ribosome to the translocase
  5. The Sec61 pore opens. The SRP is not bound to the ribosome and translation now resumes. This provides the energy to push the protein through into the pore in the ER membrane into the ER lumen (translocation)
  6. Signal peptidase cleaves the signal sequence
44
Q

Function of the Sec61 seam

A

The signal sequence binds to Sec61 due to the SRP and SRP receptor, and the central pore of Sec61 opens. Then, signal peptidase cleaves the signal sequence as the protein enters, and translation continues to push the protein through, into the ER lumen. At this point, the lateral seam of Sec61 opens, which allows for the lateral diffusion of the cleaved signal sequence to the hydrophobic interior of the membrane. The signal sequence will then be degraded, which is the main function of the Sec61 seam.

45
Q

Why must the translation of a protein by blocked by SRP?

A

The pause likely gives the ribosome enough time to bind to the ER membrane before the translation of the protein is completed, so the protein is not released into the cytosol. This is important for lysosomal hydrolases, which could damage the cell. It also makes sure that the parts of the protein that could fold are not made before reaching the ER membrane

46
Q

Sec61 complex

A

In eukaryotes, there are 4 Sec61 complexes which make up a large complex of translocators. 3 of the complexes are inactive, and just serve as binding sites for ribosomes and chaperones. 1 will serve as the actual translocase through which the protein will travel

47
Q

Post-translational protein secretion

A

Some proteins are secreted following translation. Chaperone proteins associate with polypeptides and prevent them from misfolding before they are fully secreted. We can’t rely on ribosomes to push the protein through the ER membrane during post-translational secretion. In prokaryotes, SecA ATPase is responsible for this. In eukaryotes, BiP protein

48
Q

SecA ATPase

A

A eukaryotic enzyme that attaches to the cytosolic side of the translocator in the cell membrane (prokaryotes don’t have an ER). Energy is provided through ATP hydrolysis, which allows the ATPase to go through cyclic conformational changes. The conformational changes create a “ratcheting” mechanism that pushes the protein across the membrane, through the Sec pore

49
Q

BiP protein

A

Located on the lumenal side of the ER in eukaryotes. It goes through repeated conformational changes, pulling the protein through the membrane and into the ER. It gains energy through ATP hydrolysis. Associated with the Sec 63, 71, 72 complex

50
Q

Sec 63, 71, 72 complex

A

Located on the cytosolic side of the ER in eukaryotes. It is associated with Sec61 and helps with post-translational translocation

51
Q

SecA ATPase function

A

The ATPase goes through repeated rounds of ATP binding, hydrolysis, and release (of ADP). Each of these 3 actions causes conformational changes in the protein and pushes the unfolded protein across the membrane, creating a ratcheting motion

52
Q

Function of the BiP protein

A

BiP is located in the ER lumen. It will go through repeated conformational changes due to ATP binding, hydrolysis, and release. The conformational changes create a pulling motion which pulls the protein into the ER lumen

53
Q

What makes integral membrane proteins different from other proteins?

A

Membrane proteins must have some hydrophobic portion, as the protein will be stuck in the hydrophobic interior of the membrane

54
Q

Start-transfer signal

A

Signals for the start of translocation through the pore. It serves this purpose in all translocated proteins. The signal is hydrophobic. The protein is translocated until the stop-transfer sequence is encountered. It will be cleaved off my signal peptidase

55
Q

Stop-transfer signal

A

A second hydrophobic sequence that signals translocation to stop, and translocation into the lumen stops for a period of time. This sequence anchors the protein in the membrane after the start-transfer signal has been cleaved off and released from the translocator. Translation can continue out in the cytoplasm, but the protein will not continue to be pushed through the membrane. The stop-transfer signal sequence is laterally transferred into the bilayer when the Sec61 pore opens. Since the stop-transfer sequence is internal to the protein (inside the membrane) it is what holds the protein in place in the membrane. This process creates a single-pass transmembrane protein

56
Q

Single-pass transmembrane protein

A

A protein that passes the membrane once- it is anchored to the membrane by the lateral transfer of the stop-transfer signal into the bilayer

57
Q

Double pass proteins

A

These proteins do not have a signal sequence at the N terminus, as it’s internal. This means the start-transfer sequence will not be cleaved by signal peptidase, but the sequence will still direct the protein to the Sec machinery. The start-transfer sequence binds to SecYEG and directs the protein across the pore so translocation to begin. Translocation stops once the stop-transfer sequence is encountered, but the rest of the protein is made on the other side of the membrane. Once the whole membrane has been made, the lateral seam of Sec61 opens, and both the start and stop transfer sequences laterally diffuse into the hydrophobic membrane. Both of the start and stop sequences are internal and part of the membrane, which creates a double pass protein

58
Q

Multipass transmembrane protein

A

Translocation begins with the start transfer sequence and stops at the stop transfer sequence, and the protein continues to be made on the other side of the membrane. The sequences continue to move laterally through the Sec61 machinery. The protein contains additional start/stop sequences which cause the process to begin again. All of the sequences will move into the hydrophobic interior of the membrane, creating a multipass membrane protein

59
Q

Glycosylation

A

This is the addition of 1 or more sugars to a molecule. Proteins with sugars added to them are called glycoproteins, lipids with sugars added to them are called glycolipids

60
Q

N-linked (Asn-linked) glycosylation

A

A protein modification that occurs only in the ER lumen. A precursor oligosaccharide (sugar) is added to the protein- the entire molecule is added all at once. The precursor oligosaccharide is composed of N-acetylglucosamine, mannose, and glucose. It is transferred to the amino group (NH2) on the side chain of asparagine (Asn), hence the name of N-linked glycosylation. A lot of sugars will be trimmed off in the ER and the Golgi, leaving a small piece of the original oligosaccharide remaining. N-linked is the most common type of glycosylation and is found on 90% of glycoproteins in mammalian cells

61
Q

Oligosaccharyl transferase

A

An enzyme in the membrane of the ER which its active site on the lumenal side. It catalyzes the addition of an oligosaccharide to a protein (N-linked glycosylation). When proteins with an asparagine residue enter the ER, oligosaccharyl transferase transfers an oligosaccharide from dolichol to the Asn residue on the protein. There can be more than one glycosylation is there are multiple Asn residues

62
Q

Dolichol

A

A specialized lipid in the ER membrane. It anchors the precursor oligosaccharide on the lumenal side through a high energy phosphate bond before it’s added to proteins. This high energy bond provides energy for glycosylation/moving the oligosaccharide. Precursor oligosaccharides are all pre-made, and they hang out connected to dolichol, which anchors them to the membrane

63
Q

Glycosylation of cytosolic proteins

A

N-linked glycosylation does not occur in cytosolic proteins, as they have a simpler glycosylation process. 1 N-acetylglucosamine is added to serine or threonine

64
Q

Functions of N-linked glycosylation (4)

A

Glycosylation acts as a signal to mark the state of protein folding. 1. Glucose molecules will be trimmed off in the ER to indicate if the protein is properly folded
2. Calnexin and calreticulin only bind to proteins with 1 glucose and guide the correct protein folding. This remaining glucose serves as a signal that the protein needs help folding
3. A protein is ready to leave the ER once glucosidase removes its remaining glucose, indicating the protein has been properly folded
4. If the protein still is not properly folded, glucosyl transferase adds back a glucose molecule. The cycle continues until the protein is folded properly

65
Q

Calnexin and calreticulin

A

ER associated chaperones that require calcium to function. They are lectin binding and bind to proteins with one molecule of glucose. These chaperones will guide the correct folding of the protein

66
Q

Glucosidase

A

Removes remaining glucose from a glycosylated protein, signaling when the protein has been properly folded. At this point, the protein is ready to leave the ER

67
Q

Glucosyl transferase

A

An ER enzyme that recognizes if a protein is still improperly folded after glucosidase has removed a glucose. If the protein still has not folded correctly, glucosyl transferase will add back a glucose

68
Q

ER retrotranslocation

A

Occurs when proteins have gone through multiple rounds of chaperone assisted folding and they still haven’t folded properly. How this process is recognized and mediated is unclear. The protein will be translocated back out into the cytoplasm. Once this happens, N-glycanase removes the entire oligosaccharide. Ubiquitin tags are added and the protein is sent to the proteasome to be degraded

69
Q

Unfolded protein response

A

Triggered when misfolded proteins accumulate to high levels in ER lumen

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