Chapter 7 Flashcards
Why do cells depend on signals on proteins?
to ensure they arrive at their proper subcellular destination
Proteins with NO targeting signal will be translated entirely on and remain in?
free ribosomes and remain in cytosol
Which organelles receive proteins from the cytosol after their translation is complete?
mitochondria, nucleus, chloroplasts, and peroxisomes
What are translated by ribosomes attached to the ER and are translocated at the same time?
ER, Golgi, lysosomes, plasma membrane, and secreted proteins
ER, Golgi, lysosomes, and plasma membranes, and secreted proteins arrive at the other locations via
vesicle budding and fusing
Secretory pathway
transport of proteins through nucleus, ER, Golgi, and to the membrane
Lumen of organelles of the secretory pathway are topologically equivalent to
each other and to the exterior of the cell
Proteins targeting selectively recognizes
nascent proteins with signal sequences
The signal sequence binds to
signal recognition particle
Binding of signal sequence to the signal recognition particle causes
translation to temporarily halt
Which 3 arrive at the ER
ribosome, nascent polypeptide, SRP
What happens when the ribosome, nascent polypeptide, SRP interact with a receptor at the ER?
a channel (part of the translocon) opens and the nascent proteins begins to be co-translationally translocated
When was the signal hypothesis proposed?
1970
Why was the signal hypothesis proposed?
to explain how proteins got targeted to the RER
When is the ER targeting signal cut off?
after the protein begins its translcoation
Pre-protein
nascent protein prior to its signal sequence removal
Normally ER bound protein synthesized in an in vitro cell-free system for gel
larger, migrates more slowly
When translated in vitro in the presence of purified ER (microsomes) for gel
protein smaller, migrates faster, located in ER lumen (microsome lumen)
What particle is SRP?
ribonucleoprotein
SRP made up of
6 polypeptides and small (7S) RNA molecule
What of SRP has what activity?
GTPase activity
The part of SRP that binds to the nascent polypetide’s signal sequence has a
large number of Met residues (hydrophobic)
function of part of SRP that binds to the ribosome
slows translation until docking at the ER
Part of SRP bins to what? (2)
a. nascent polypeptide’s signal sequence
b. ribosome
SRP receptor structure
Dimer of two subunits: SRalpha and SRbeta
Where are SRalpha and SRbeta?
SRalpha - on cystol face
SRbeta - transmembrane
SRalpha and SRbeta are what proteins?
GTP-binding porteins (GTPases)
Coordinated GTP binding and hydrolysis by SRP and SR are required for
a. proper targeting of nascent chains to the ER
b. for their transfer to the translocation channel
c. for the recycling of SRP to the cytosol
What happens when SRP releases the ribosome?
Ribosome engages the translocon and the nascent polypeptide begins translocation
Transport of the polypeptide into the ER lumen through
an aqueous channel
Translocon comprised of
channel and other proteins closely associated with it
Ions cannot cross the membrane while
polypeptide is being translocated through a channel
After polypeptide is released and the ribosome is still attached, ions
permeate through the channel
Sec61 function
forms the channel through which the translocating protein passes
Sec61 structure
heterotrimeric complex, shaped like an hourglass in cross-section
What commits the chain to translocation?
Recognition and insertion of the signal sequence
What is displaced as translocation begins?
channel plug
During translocation, some proteins are able to
transiently slip out of the gap between ribosome and translocon, positioning a loop of the protein in the cytosol
Some organisms (especially unicellular eukaryotes) are able to translate and translocate proteins in where?
translation in cytosol, keep them unfolded, and translocate them into ER
Protein is no longer associated with ribosome when
it is translocated
Chaperonins of the hsp70 family associate with
nascent polypeptides
Chaperonin of the hsp70 family function
prevent holding
What is most important for recognition by the channel?
hydrophobicity of the signal sequence
What is the main energy source driving posttranslational translocation and co-translational translocation?
ATP hydrolysis by the ER-lumenal hsp70 BiP protein
What does the active pulling model propose?
ATP hydrolysis causes a conformational change in BiP which causes the polypeptide to be actively pulled (or pushed with SecAp for prokaryotes) through the channel
Steps of polypeptide translocation using BiP
- BiP interacts with Sec63 and binds the polypeptide
- polypeptide can diffuse inwards (ER lumen), but BiP prevents backward diffusion
- when enough polypeptide is exposed, a second BiP binds
- diffusion occurs again
- process repeats, advancing the polypeptide incrementally
What side is the N-terminus on?
on the cytosol face or on the non cytosol face
What side is the C-terminus on?
on the opposite face from the N-terminus or may be on the same face
Steps of protein translocation (N-terminus)
- protein translocation begins with signal sequence at the N-terminus
- channel recognizes transmembrane domain
- transmembrane domain enters the lipid bilayer through side of the channel
- translation continues until termination
- integrated polypeptide
Signal anchor proteins target by using an
internal transmembrane domain (signal anchor)
Simplest situation of transmembrane orientation
proteins spans the membrane 1x, the N-terminus faces ER lumen, and C-terminus faces the cytosol
Two possible orientation?
N-terminus region translocated into the ER-lumen or the C-terminus translocated into the ER-lumen
Polytopic proteins
span the membrane multiples times; may have transmembrane regions integrate one-at-a-time or they may integrate in pairs
If the ER-signal peptide is ________, it is almost always..?
N-terminal; cleaved after it has served its purpose
Signal peptidase complex structure
5 subunits: 2 of which have proteolytic activity
Exact cleavage site is?
variable
Exact cleavage site is influenced by the
amino acid residues in the immediate vicinity of the cleavage sites
Following its removal, signal peptide is often processed by
signal peptide peptidase
GPI anchoring
lipid GPI added to some translocated proteins
GPI anchoring to proteins renders them attached to the membrane as
IMPs
IMPs
integral membrane proteins, non-removable from the membrane by salt extraction procedure
GPI anchoring always tethers a protein to the
non-c-face of a membrane (ER-lumenal face first)
GPI
glycosylphophatidylinositol
Where does GPI begin at and move to?
begins on the c-face of the ER membrane, then a flippase (translocase) moves it across to the lumenal side
What happens to GPI at the lumenal side
more sugars are added along with 3 phosphoethanolamines
Signal for GPI-anchoring
small C-terminal hydrophobic domain of variable length
What recognizes the signal for GPI anchoring?
integral membrane complex
Integral membrane complex function
cuts the signal off from the protein and transiently attached to new C-terminus (omega site) and attaches omega site to the terminal phosphoethanolamine residue of GPI and the enxyme complex liberates itself
3 reasons why GPI-anchoring occurs
- way of targeting proteins in polarized cells, lipid rafts…
- give an IMP more lateral mobility compared to transmembrane IMPs
- way to free a protein from membrane association by enzymatic cleavage of its attachment, thus passing along a message into the interior of a cell
More than half of secretory and membrane proteins in a cell are
glycosylated
Glycosylation in the ER is termed ____. Why?
N-linked because sugars are attached to asparagine residues
OST
oligosaccharyltransferase
Function of OST
transfers an oligosaccharide en bloc onto the translocating protein
Oligosaccharide presynthesized beginning in the
cytosol
Oligosaccharide presynthesized with the minor membrane ____.
phospholipid dolichol-phosphate
Sugars are added to ____.
dolichol-P
dolichol-P moved to where and by what?
flippase moves it to the lumen-face of the membrane
After oligosaccharide is added, what happens and where?
removal of some sugar residues and addition of others within the ER and Golgi
Most abundant protein in the ER lumen
BiP (binding protein)
BiP
Chaperonin
BiP most abundant in
ER lumen
BiP is a member of the
hsp70 family
BiP binds to
exposed hydrophobic patches (buried within a globular protein, interacting with each other to drive protein folding)
Why does BiP bind to exposed hydrophobic patches?
To help them get folded properly
ER-lumenal chaperonins no longer associate when?
once the protein has achieved its proper folding?
Cytosol has a __________ environment
reducing
In the reducing environment of a cytosol, what is not formed?
disulfide bonds
ER lumen and ECM have _________ environment
oxidizing
In the oxidizing environment of the ER lumen and ECM, what is formed?
disulfide bonds
The formation of the disulfide bonds in the oxidizing environment is catalyzed by
protein disulfide isomerases
Where in the cell has oxidizing environment and reducing environment?
Reducing environment - cytosol
Oxidizing environment - ER lumen and ECM
PDI
protein disulfide isomerase
PDI catalyzes
disulfide bond rearrangement and formation
PDI reduced when it
oxidizes the cysteines in a translocating protein
Reduction of PDI helps
folding
What oxidizes PDI by being itself reduced?
ER protein EroP1
PDI is oxidized by
an ER protein EroP1
EroP1 oxidized by
FAD
What is calnexin and where is it?
IMP in the ER memebrane
What is calreticulum and where is it?
protein in the ER lumen
Calnexin and calreticulin binds to
sugar residues
Binding of calnexin and calreticulin to the sugar residues indicate that they are
lectins
The purpose of calnexin or calreticulin activity is in
protein quality control (QC)
The calnexin or calreticulin activity is to
make certain that proteins are properly folded before they are allowed to leave the ER
What is the ticket to enter the calnexin cycle?
To have been N-glycosylated
After entering the calnexin cycle, the 2 distal glucose residues are removed by
glucosidase I and II
Association of calnexin helps the
protein contact ERp57
ERp57
a chaperonin and PDI family member
ERp57 function
catalyzes the disulfide bond formation and rearrangement and allows folding/refolding
What protein lets go of calnexin
unglycosylated protein
If the protein is not folded properly, a glucose is re-added by
UDP-glucose-glycoprotein glucosyl transferase (UGGT)
How does UGGT know that a protein is not folded properly?
it would be exposed clusters of hydrophobic residues
Proteins in the secretory pathway are retained in
ER by chaperone-association until the assembly of its subunits are completed
3 Retention mechanisms
a. associating a subunit with BiP
b. exposed cysteine binding to a PDI
c. exposed ER retention signal that will be masked later
BCRs or secreted Ig depend upon the
association of 4 polypeptides, 2 H chains (transmembrane) and 2 L chains (free in ER-lumen)
Retrograde translocation is used to
export misfolded proteins back into cytosol
What happens to the misfolded proteins in the cytosol?
multi-ubiquitinated and degraded by a proteasome
Ubiquitin
ubiquitous small protein used as a tag for protein degradation
Proteasome
cytoplasmic recycling barrel, a large protease complex
Degradative pathway (ubiquitinated protein degraded by proteasome) is known as
ER-associated degradation (ERAD)
ER-associated degradation (ERAD)
Misfolded proteins that cannot get refolded properly moves into the cytosol, and they are tagged with ubiquitin, marking them for degradation by proteasome
One of the retrograde translocation channels
Sec61
Unfolded protein response (UPR)
signaling pathway from the ER lumen to the nucleus
UPR function
a. allows the cell to monitor folding conditions
b. increase the expression of ER chaperonins when necessary
Protein that senses folding conditions in the ER and transmits the info
ER-membrane protein Ire1p
Ire1p has the ability to
dimerize by self-association of its lumenal domain
Under normal conditions, _______ bound to the lumenal domain of _______ resulting in?
BiP; Ire1p; inhibition of dimerization
Under stress, BiP is
too busy with misfolded proteins, so it leaves Ire1p to dimerize and send a signal
Cytosolic domain of Ire1p contains
a serine-threonine kinase
Serine-threonine kinase
it phosphorylates proteins on serine and threonine residues
When Ire1p dimerizes, it does what two things?
autophosphorylates and activates a second cytosolic domain
Dimerization of Ire1p catalyzes what?
the removal of an intron from the mRNA of a specific gene, HAC1
After Ire1p has removed HAC1, tRNA ligase does what and resulting in?
tRNA ligase joins the axon, allowing Hac1p (mRNA) to be translated
What happens if HAC1 (intron) is left in?
causes ribosomes to stall on the mRNA, causing no Hac1p to be translated
What is Hac1p?
transcription factor
What does Hac1p bind to?
a regulatory sequence called the unfolded protein response element (UPRE)
What does binding of Hac1p to UPRE stimulate?
transcription of several chaperonin genes
ex) BiP
Making the additional chaperonins help
QC with unfolded proteins during stressful conditions
Unfolded protein response (UPR) influences
transcription of a much broader collection of genes beyond those for chaperonins
What process is altered as a consequence of the cell’s UPR?
increased lipid synthesis for ER expansion
Primary site of sythesis of the cell’s phospholipids
ER
Where is the cell’s phospholipid made in?
cytosolic leaflet of the ER membrane
Kennedy pathway
two fatty acyl CoAs are attached to glycerol 3-P to form diacylglycerol (DAG), which is hydrophobic enough to insert into the c-face of the ER membrane
From the membrane-associated DAG, what group can be added to form a complete phospholipid?
polar head groups
How is the polar group added? (STEPS)
- head group phosphorylated
- attached to CDP
- Head group + a P is transferred to DAG
- Releases CMP
What is the most abundant membrane phospholipid?
Phosphatidylcholine
What 4 membrane phospholipid is made by Kennedy pathway?
Phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PL), phosphatidylinositol (PI)
The 4 membrane phospholipid (PS,PE, PC, PL) is made by the Kennedy pathway in the ?
cytosol-face of the ER
Where can PE be made other than ER?
MItochondria by modifying PS
A specialized ER region that is responsible for transport of PS from ER
mitochondrial-associated membrane
What is mitochondrial-associated membrane (MAM)?
specialized ER that is very close to a mitochondria, making very close contact with its membrane
What synthesis also occur in the ER?
Sterol (most cholesterol)
Following their synthesis in the ER, some phospholipids must be transferred to where?
all the other membranes in the cell and NOT randomly
Transferring phospholipid to other membranes may occur in which regions?
where ER makes direct contact with other organelles of the cell
Phospholipid transfer proteins thout to move lipids from one bilayer to another but they cannot?
account for membrane growing because after they bring a lipid to membrane, they leave with a different one
Flippases neceessary to?
flip half of the newly-synthesized lipids from ER c-face to the non-c-face
Flipping of the lipid occurs randomly, but?
other phospholipid translocators must be more selective because memrbanes in cells are varied in their lipid composition from each other and within any membrane the two faces are varied
Where are PC, PS, PE, and PI enriched?
PC in non-c-face of the plasma membrane
PS and PE in the c-face
PI in c-face (EXCEPT GPI-anchored proteins)
ER form morphologically and in other regions
Morphologically, ER forms large, flat sheets (cisternae)
In other regions, it is in form of long, curving tubules
The flat sheets are generally found next to the
nuclear envelope
Tubules extend as a
network throughout the cell, contracting other organelles and the plasma membrane
RER is abundant in cells which secrete ?
proteins like Ig (plasma B cell), hormones,,
SER responsible for
lipid metabolism steroid synthesis, glycogen metabolism, and drug detoxification
SER is abundant in cells which secrete
steroid hormones (testes’ leydig cells, ovary follicular cells) or liver hepatocytes
SER amount goes up with an?
increase in drug use
SR in skeletal muscle cells, cntain
calsequestrin
What is calsequestrin?
protein that has several Ca2+ binding sites
SR stores
intracellular Ca2+
SR in other cell types enables?
rapid Ca2+ regulation
What is calreticulin?
glycoprotein chaperone and a Ca2+ binding protein
When does BIP, calnexin, and calreticulin functio less efficiently?
when ER Ca2+ stores are depleted
Prolonged calcium depletion can induce?
UPR
Ca2+ release from the ER is implicated in?
triggering apoptosis
Tubular elements of the ER are in
continual flux, aligned to the cytoskeleton
Cytoskeleton is not required for the
formation of tubules or networks in vitro
Role of cytoskeleton ensures that
after the ER network forms, it is properly distributed throughout the cell
In response to increase in what drug expands SER?
phenobarbital
Removal of phenobarbital causes
rapid return of the SER to its normal size
The plasma B cells proliferate their RER in order to
meet the need for the cell to secrete large amounts of Ig proteins
Nucleus uses
internal signal patches
Internal signal patches are comprised of
non-contiguous aa residues that are brought together by tertiary and quaternary structure
What is not removed after import into the nucleus through the nuclear pore complexes?
nuclear localization signals (NLSs)
Why are NLSs not removed after import into the nucleus through the NPCs?
because many of the proteins might need their signal if they are outside after telophase and it is difficult to remove internal signals
Nucleus is able to import proteins in what form dues to what?
folded form due to size of the NPCs
Mitochondria and chloroplasts must import proteins in what form?
unfolded form
Peroxisome import proteins in what form?
folded form
Organelle targeting signals are
N-terminal or C-terminal
When are organelle targeting signals removed?
post-translocation
What spans the outer mitochondrial memebrane?
Translocase of the outer membrane (TOM)
Each mitochondrial membrane is spanned by a
different multi-protein complex that includes translocation channel to import proteins
What spans the inner mitochondrial membrane?
Translocase of the inner membrane (TIM)
What is the mitochondria targeting signal?
An N-terminal amphipathic alpha-helical stretch of 20-55 aa residues with hydrophobic sequences on ‘side’ of the helix and basic aa residues on the other
Why is chaperone cytosolic hsp70 family necessary?
To prevent folding of the protein prior to translocation
Portions of what are in close proximity?
TIM and TOM
Why are TIM and TOM in close proximity?
to ensure that most proteins are passed between complexes without dissociating into the intermembrane space
What binds protein as it emerges from the channel
mtHsp70
mtHsp70 act as what 2?
ratchet or motor or both
mtHSp70 binding to protein to act as a ratchet or motor requires
ATP energy
Membrane potential across the inner membrane (ETS) is important in
translocation
Most mitochondrial signal sequences are cleaved by
soluble mitochondrial processing protease (MPP)
One way that proteins arrive at final location of transmembrane IMPs in the mitochondrial inner membrane is via
Oxa1p pathway
What happens in the Ox1p pathway?
Proteins from cytosol get into the mitochondrial matrix, have their mitochondrial signal removed, exposing a second signal with directs them to the membrane for insertion
Proteins translated within the mitochondrial matrix could arrive at that destination with a
single targeting sequence
Proteins destined for chloroplast are kept unfolded in the cytoplasm by
cytoplasmic hsp70s
Channel that preproteins destined to chloroplast first meet?
Translocon of the outer envelope of chloroplasts (TOC)
Channel that proteins destined to chloroplasts go through and is attached to the TOC
translocon of the inner envelope of chloroplasts (TIC)
What requires energy for the proteins to go to the chloroplasts?
chloroplast stroma hsp70-type of chaperone
What regions of the chloroplasts must receive their own subset of specific proteins?
Thylakoid space and thylakoid membranes
What translocation system to thylakoid proteins use?
Twin-arginine-translocation system (tat)
TAT system uses what signal sequence?
second signal sequence since used after the chloroplast-targeting signal is cleaved
Translocation across the thylakoid membrane requires what 2?
energy and pH gradient produced during photosynthesis
What signal sequences can be used to import proteins into peroxisomes?
C-temrinal (PTS1) or N-terminal (PTS2)
Proteins are imported into the peroxisomal matrix by
posttranslation and post folding
Receptor that is carried into the peroxisome and then is exported alone to be used again
Peroxisomal targeting signal receptor
Why do peroxisomal translocation channel has to be tightly regulated?
the internal environment of peroxisomes would be toxic to the cell if the peroxisomal membrane were as permeable to protein and small molecule movement as is the nuclear envelope
What are the two main transport routes?
exocytic pathway (secretory pathway) and endocytic pathway
New proteins destined for any locations within the endocytic/exocytic pathway must be first targeted to where?
ER
What are the two ways to exit the ER?
a. to fail to fold properly, retrograde transport out to be ubiquitinated and proteasomed
b. to exit via budding into a transport vesicle
What compartments form during endocytosis?
Endosomes
Transport has to be ___________ lest the donor compartments cease to exist
bi-directional
What mechanisms return some vesicle components to the donor compartments?
Recycling mechanisms
What mechanism returns resident proteins which escaped from their donor compartments?
Salvage mechanisms
When is pulse-chase experiment used?
To experimentally show the pathway that proteins take as they move through the secretory pathway
What is regulated selection?
when cells accumulate proteins to be secreted in vesicles near the plasma membrane releasing them upon stimulation
Regulated secretion is aka
inducible secretion
What are 3 examples of regulated secretion?
a. digestive enzymes (pepsinogen, trypsinogen)
b. hormones (insulin, ADH)
c. histamine
What is constitutive secretion?
when cells continuously secrete a protein
What 4 examples of constitutive secretion?
a. immunoglobulins
b. yolk protein
c. bacterial infection-promoting proteins
d. insulin
What compartment is the most abundant membrane in most eukaryotic cells?
ER
What is Golgi apparatus made of?
Golgi stack made of cis, medial, and trans cisternae
What part of Golgi is near the entry face and what part is nearest the exit face?
cis-Golgi near the entry face
trans-Golgi near the exit face
Within the Golgi stack, what are modified sequentially to highly sialylated structures?
high mannose oligosaccharides (added to proteins in the ER)
What is the function of cis-Golgi network (CGN)?
a. receive proteins from the ER export sites
b. in QC by allowing ER resident proteins that escaped to be returned
Trans-Golgi network sorts for distribution to what 3 places?
a. lysosomes
b. plasma membrane/constitutively secreted proteins
c. regulated secretion
What are the 4 endocytosis functions?
a. internalizing of nutrients
b. regulating the cell surface expression of receptors, transporters
c. uptake, recycling of EC debris
d. recovery of membrane from the plasma membrane
What is the range from least to most degradative?
early endosome, late endosome, lysosome
What are V-ATPases?
H+ ATPases that transport protons from cytosol into the organelle lumen
Why is the acidification of early endosomes important?
for the dissociation of internalized ligand receptor complexes and recycling of cell membrane receptors to the plasma membrane
Why is the acidification of late endosomes important?
for the delivery of lysosomal enzymes from the trans-Golgi network (TGN)
What 2 cells are professional phagocytes?
macrophages and dendritic cells
What do phagocytes do?
ingest pathogens and clear senescent or apoptotic cells via phagocytosis
RME
Receptor-mediated endocytosis
Where does transcytosis occur?
in epithelial cells lining the intestine and other body cavities
What does transcytosis allow? (2)
a. nutrient uptake via the formation of a clathrin-coated vesicle
b. allow infants to take Ig from mother’s milk by binding the Ig to gut apical receptors, transferring Ig through to the other side, and releasing the Ig into the blood plasma
