What are two ribosome cycles?
Free cytosolic and ER bound
How can proteins be delievered to the ER
Via post or co translational mechanism
What is SRP
Signal recognition particle. That binds the sig seq on the new polypeptide. Once it leaves ribosome translation is paused on ribosome and drags the whole complex to ER
Co-translational steps
1. SRP binds to the Sig seq as soon as peptide is released from ribosome.
2. This causes a pause in translation and allows the ribosome to bind to the ER membrane, before synthesis is completed ensuring that the protein is not released into the cytosolic.
Compare co and post translational translocation
In co translocation- translocation is driven by translation
In post translocation- ATP is req to feed protein through pore
Microsomes
Small vesicle derived from fragmented ER produced when cells are homogenized. Microsomes form from disrupted ER, have same topology and fxn as ER .Aka isolation of rough ER useful for studying the import of proteins into ER . (Can get smooth and rough mic
Signal hypothesis
Proteins destined for secretion, which involves the movement of protein across a biological membrane, are originally manufactured with an initial seq of aa that May or may not be present in the mature protein
Integration of a single pass transmembrane protein into the ER membrane
It's all about the stop transfer seq. when the stop transfer seq enters the the translocon and interacts with a binding site, the translocation changes it's conformation and discharges the protein into the lipid bilayer laterally
Integral membrane proteins
are membrane proteins whose final destination is the plasma membrane. they are translated into er and move into membrane co-translationally. but into a vesicle from er to the golgi to the plasma membrane and fuse into membrane at the cell surface.
hydropathy index
measure of an amino acids hydrophobicity. useful for determining whether a protein has a transmembrane domain.
transmembrane membrane
part of the protein that is likely to go through the membrane, hydrophobic. polar proteins wont be in transmembrane domain, usually are hyrophobic non polar protein (alpha helices ). the hydrophillic portion will stick out at end. (transmembrane proteins
is integral membrane protein topology maintained?
go over topology again (lecture 3)
yes, its important to maintain topology to smooth transition. topology is maintained throughout the secretory pathway.
An internal signal sequence can be used to integrate a single-pass transmembrane protein into the ER membrane in two different ways (internal seq in this case acts as a stop seq)
In one case, the resulting membrane protein has its C-terminus on the lumenal side (Figure 12-48A), while in the other, it has its N-terminus on the lumenal side (Figure 12-48B). The orientation of the start-transfer sequence depends on the distribution o
How can you follow proteins through the secretory pathway?
Pulse chase exp: Using labeled amino acids to follow proteins through the secretory pathway . Other methods could be western blot, GFP etc.
In order to go to ER you MUST have sig seq
can send almost anything to ER as long as you ave sig seq. Sig seq are most likely conserved
Internal anchor seq
(Start transfer/ internal signal sequence - signal sequence that also acts as a transmembrane domain - binds SRP and translocon)
internal sig seq can also act as a stop transfer in some cases, aka acting like a transmembrane domain thus there would be no
remember internal transmembrane domain is NOT cleaved. only cleavage occurs at the ends (@terminals)
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what is the transmembrane domain
is the part of the protein that goes through the plasma membrane. the membrane is lipid so only hydrophobic things will be happy there, so needs to be 20-30 aa to span the membrane.
Insertion of a double pass transmembrane protein with an internal signal sequence
insertion of a double pass transmembrane protein with an internal signal sequence is not cleaved .
also Insertion of a multipass transmembrane protein into the ER membrane does not have its signal sequence cleaved because it is located internally.
thus bo
GPI anchor protein? destination?
happens in the ER, where transmembrane domain that will anchor protein into ER, THE GPI anchor will get transferred and cleaved.
destination is plasma membrane
What happens once the protein is in the ER?
Disulfide bond formation - PDI
Folding - chaperones, peptidyl prolyl isomerase
Addition and processing of carbohydrates (also in Golgi)
Proteolytic cleavage (also in Golgi)
Assembly into multisubunit proteins
how to polypeptides fold ?
chaperones (Hsp70 family- heat shock) help it fold, binds to hydrophobic regions to prevent it from aggregating to give it time to fold.
disulfide bonds
oxidative linkage btwn adjacent cysteines, that stabilize the protein structure, particularly in 3 and 4 structures, happens in ER cuz its more oxidizing so better environment than cytosol. the reducing environment in cytosol usually prevents disulfide bo
PDI (protein disulfide isomerase)
catalyzes the rearrangement of the disulfide bonds. by making disulfide bonds with the protein itself.
Antibody (Disulfide bond example)
made of protein chains connected by disulfide bonds
peptidyl propyl isomerase
rotate aa around the peptide bonds, to get protein into right conformation for folding
glycosylation of proteins in ER
depending on whether you have one sugar conformation over another will determine whether calnexin will bind to the protein and retain it in the ER. calnexin/calreticulum binds protein and keeps it in the ER until it can fol
impt for quality control.: such as protein folding* (not a major req, but w/o it the protein will degrade quicker, so is considered a protein marker), stability (particularyly in asparagine) , cell-to-cell adhesion
*the presence of oligosaccharides tends
question: Therapeutic proteins such as antibodies, tissue plasminogen activator (anticlotting factor) and erythropoietin (stimulates production of red blood cells)used to be mass produced in bacteria. This process was very inefficient and expensive, becau
These proteins needs to fold properly but without an ER, you will not fold properly because wont have glycosylation,
so bcuz bacteria don't have ER the therapeutic proteins won't fold correctly
thus by changing the host to mammalian cells which have an ER
quality control
ERAD- ER associated degrada tion
UPR- unfolded protein response
both happen under diff circumstance
only properly folded and assembled proteins can leave ER
If protein is not properly folded they are removed by ERAD (Irreversibly misfolded proteins are retrotranslocated, ubiquitinated, and degraded by the proteasome)
an excess of unfolded proteins in ER will activate UPR
ER chaperones (primarily assist in proper folding), peptidyl-proyl isomerase & protein disulfide isomerase
assist in proper folding, increase transcription of proteins that assist in proper folding.
calnexin & calreticulin (retaining folded protein) bind to sugar (carbohydrates)
bind N-linked oligosaccharide(sugar (oligosaccharide) and retain protein in ER until properly folded
calnexin- membrane protein in ER , hold onto carbohydrates to retain it in the ER while its folded
calreticulin-fxn in retaining protein in ER until prope
protein gets out ER
enzymes are trimming the sugars during folding process,
enzyme in ER will cleave and adding sugars which is regulating the adding and removal of calnexin.
3 glucoses (SUGAR): an enzyme will trim 2 of the glucose which will allow it to bind to calnexin all
Ubiquitin (assists getting misfolded protein to ERAD)
small protein, covalently added to other proteins, present in eukaryotes, added to lysine
have 3 enzymes
E1 (activates),E2, E3- e3 will bind to substrate that needs to be degrades, . if something is wrong with E3 than will get lots of protein that won't b
UPR (excess of unfolded protein in ER)- fxn to increase expression of chaperone
1.Increases transcription of proteins that assist in proper folding
2.ER chaperones, peptidyl-prolyl isomerase, and protein disulfide isomerase
The excess of unfolded proteins in ER will activate the transmembrane kinase in ER
Will activate endoribonuclease (cleaves out RNA)domain within the kinase- which will splice out an intron from the RNA in the nucleus that will encode a transcription facto
Proteins in ER membrane sense accumulation of misfolded proteins, signals to nucleus to activate transcription of genes involved in protein folding
sensors are on the inside of the lumen of ER. misfolded proteins in ER signal the need for more chaperones. once misfolded protein is sensed, activation and phosphorylation of sensor occur which will activate the endoribonuclease (UPR) so it can splice ou
Protein folding in ER (OVERVIEW)
1.Chaperones (fold proteins)
2.Disulfide bonds - PDI (get it in proper orientation to fold)
3.Rotation around peptide bonds - peptidyl-prolyl isomerases
4. Glycosylation
5. ERAD (degrade misfolded proteins)
how do proteins move between compartment in the secretory pathway ?
vesicular transport- budding in a vesicle
proteins are always in vesicle, never enter cytoplasm
protein coat (such as clathrin, COPI,COP11)
without protein coat there is no vesicle formation
PURPOSE: COPP11
1.concentrate certain proteins in region that will give rise to vesicle
2.assembly into curved basket type lattice promotes shape of vesicle
CLATHRIN
clathrin will be the coat helping with formation of vesicle
adaptin will bind to clathrin & rec proteins which will bind cargo thus concentrating everything for the vesicle creating the bud formation.
dynamin will mediate "pinching off" of clathrin coated
SNARES
are involved in membrane fusion and force water out
snares must disscoiate so they can recycle and participate in another round of vesicle transport
Question lec 5
You are studying the UPR and you believe you have found a new/novel transmembrane kinase that senses and responds to misfolded proteins. To convince your boss that this protein is worth studying, you deside to do one experiment to provide e
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question lecture 5
You are studying a protein that appears to be a member of the Hsp70 family of chaperones. You believe it is an ER resident chaperone. You look at it using immunofluorescence using a BiP antibody (green) and get the following result.
1.KDEL signal is at the end
2. Cut off KDEL and see if the protein returns to the ER. OR
Take retrieval sig and place it on another protein not originally destined for ER and see if it knows where to go (ER in this case). A secondary exp would be to see i
question lec 5 youtube
proteins move between compartments in transport vesicles. explain the basic mechanism behind vesicular transport.
make sure to include cargo packaging, vesicle formation and release, and fusion with acceptor compartment
what would b
concentrate the cargo in the ER into a certain location, adaptin will bind to rec and clathrin or w.e coat will bind to adaptin and help with curvature of vesicle. dyenmin will help with pinching off with vesicle. than snares mediate membrane fusion.
if t
GTP binding proteins
are molecular switches that usually activate
they are controlled by GAPS (GTPase activating proteins) and GEFS (guanine nucleotide exchange factors)
Rab proteins- help determine the specificity of vesicle targeting
so i Rab binds to GDP -its inactive
if R
Difference between COP I and COPII
* is that cop1 vesicles are leaving the cis golgi and moving towards the ER, this is known as retrieval transport aka retrograde transport.
*cop2 coated vesicles are budding of from the ER and encountering other vesicles. They end up fusing and forming a
What is KDEL? What is its function? How does the KDEL receptor know to release the proteins in the ER and not in the Golgi or vesicular tubular cluster?
What if KDEL is missing? Other mechanism for retaining ER or Golgi resident proteins
*sometimes proteins that are meant to stay in the ER escape to the golgi.
*Proteins that belong in the ER have a particular seq associated with them called KDEL (lysine, aspartic acid, glutamate, leucine)
KDEL rec will bind to ER proteins and bring them b
What happens in golgi?
*Further trimming and addition of sugars (n-linked oligosaccharides ) (side note remember sugars were trimme dwith calnexin in the ER)
*O-linked glycosylation (Added to OH of serine and threonine)
Proteoglycans
*Sulfation of glycosaminoglycans
AKA additio
Why glycosylated proteins?
*Glycosylated proteins bcuz help limit interactions with other proteins (space of proteins on ell surface with other proteins on cell surface),
*protein folding,
* cell adhesion and
*regulation of cell signaling
Ex of glycosylation function: Lectin bind t
How do we know that different enzymes are present in different Golgi compartments?
They did staining which showed enzymes in different compartments of the golgi
lysosome
*site for ubiquitin independent degradation or digestion.
*contain active enzymes under acidic conidtions (ph~5)
*are acid hydrolases because only active at an acidic ph
(acid hydrolases work in the lysosome so they are not degraded)
How does this protect
endosomes
Endocytic pathway - endosomes ultimately become lysosomes
delievery of material to the lysososme for digestion
through:
*phagocytosis
*endocytosis
*autophagy
How does the Golgi know to add M6P?
sig path
Golgi knows how to add m6p due to signal patch in the lysosomal hydrolase recognized by glnac phosphotransferase
How do lysosomal proteins get from the Golgi to lysosome? M6P
*lysosomal proteins are tagged with Mannose-6-phosphate
*Why are they tagged with M6P?
only sig patch for lysosomal proteins are recognized by M6P
*How does this target lysosomal proteins go to the late endosome (and then lysosome)?
*What other component
How does the M6P receptor know to recycle
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The lysosome is not always the end of the line
Lysosomal secretion
*Removal of indigestible debris (under stress)
*Specialized cells use this process under normal conditions
Example - Melanocytes - produce and store pigments in lysosomes = melanosomes
Release pigment into extracellular space
Pigment t
what is an endosome?
*endosomes can be transported from the trans-Golgi network and either continue to lysosomes or recycle back to the Golgi.
*Endosomes provide an environment for material to be sorted before it reaches the degradative lysosome
*Early endosomes then mature i
endocytosis types:
phagoctosis
pinocytosis:
-clathrin coated vesicles
-cavaloe
*phagocytosis- ingestion of large particles,
*pinocytosis- ingestion of fluid and solutes via small pinocytic vesicles
*clathrin coated vesicles- invaginate if they are clathrin coated
*cavaloe- invaginate based on lip composition of membrane (rather than
receptor mediated endocytosis
specificty to bringin exactly what you need,
macromolecules bind to complementary transmembrane receptor proteins, accumulate in coated pits, and then enter the cell as receptor-macromolecule complexes in clathrin-coated vesicles (see Figure 13-41). Recep
Why receptor mediated endocytosis instead of pinnocytosis or other non-receptor endocytosis?
What if there were a mutation in the gene encoding clathrin?
What if there where a mutation in the genes encoding the proton pump in the late endosome? Lysosome?
*specificity to bringing exactly what you need,
*if a mutation in clathrin than there will be no vesicle formation
*than ligand and rec will dissociate and not function.
than the ph will change (less acidic)hydroyslases wouldnt fxn in lysosome
LDL would n
HIV Nef downregulates CD4 by inducing endocytosis of the HIV receptor
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A signal sequence determines recruitment of a plasma membrane protein into clathrin coated pits for endocytosis
lec 8 question
You are studying a new receptor
You are trying to determine whether it participates in receptor mediated endocytosis through clathrin coated pits
You know the protein's amino acid sequence
How can you use recombinant DNA technology to addre
*look for an endocytosis signal 1st
*remove the endocyotisis signal and see if you still get endocytosis
*add endocytosis signal to something else and see if you still get endocytosis
*can use PCR to seq the protein of interest
Unless cargo is diverted elsewhere, the default pathway for molecules taken in through endocytosis is the lysosome
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fate of lysosome receptors
*Recycling of receptors to the same plasma membrane (ex: LDL rec
*Transcytosis - transit of receptors to a different plasma membrane (in polarized cells)
*Degradation in the lysosome
-another ex of recycling rec is transferrin
Why is this a economical way
receptor down regulation through endosomes
beneficial for if you want to stop the proliferation of a protein. thus send a rec. for degradation through endosome.
Why would the cell want to send the receptor to the lysosome?
To shut off a process. Shut off that signaling pathway
What is the purpose of sequestration in MVB?
Why not just send the receptor complex directly to the lysosome in an endosome? bcuz want to hide the cytoplasmic tail
Receptors can also be down-regulated through sequestration in multivesicular bodies (MVB)
the rec has been modified w/ ubiquitin & down regulated. however if the cytoplasmic tail (part of rec) is still there in the cytosol, so it can be activate other pro
endosomes can store membrane proteins for use when needed
What kinds of cells might have large intracellular pools of glucose transporters? fat cells (adipose) & muscle cells
ex: signaling through insulin rec stimulates localization of glucose transporter to the plasma membrane.
this means that endososomes can be recycyled and send signals for relocalization of the glucose rec to the plasma membrane to boost glucose uptake int
Regulated secretion vs constitutive secretion
mechanism for diversion? all signal mediated (i.e. lysosome (m6p rec) & secretory vesicle)
regulated secretion- in response to a stimulus/signal ex: insulin
constituitive secretion- always being secreted aka not being regulated. does not require a signal. default pathway for all protein leaving the cell ex: ATP , lipid ( cell division)
The cons
secretory vesicles?
What kinds of cells might have secretory vesicles?
contain specialized products that are secreted in response to a signal . than it is possible that they go back to the golgi to concentrate it , creating a mature secretory vesicle
are molecules destined for secretion are stored prior to release
Secretory
proteins go from the ER-GOLGI-PLASMA MEMBRANE or they can be diverted to LYSOSOMES OR SECRETORY VESICLES
the lumen of the trans compartment is thought to be continuous with the trans Golgi network, where proteins are segregated into different transport packages and dispatched to their final destinations�the plasma membrane, lysosomes, or secretory vesicles.
Ways of moving between different compartments
Vesicular transport - secretory pathway
Gated transport - cytosol to nucleus
Transmembrane transport - utilize protein translocators
Examples??
What factor determines where a protein goes? sig seq / tag
Transport between the nucleus and cytosol:
Why would proteins need to enter and exit the nucleus?
What kinds of proteins would you expect to enter the nucleus?
dna replication proteins & gene expression in transcription factors
Exit? RNA (mrna)
enter nucleus: proteins that affect gene expression in transcription factors
exit nucleus: rna
Macromolecules enter and exit the nucleus through nuclear pore complexes
Numbers of NPC change dependent on???
what pt of cell cycle, etc. various factors
Active transport is required for larger proteins to enter the nucleus (i.e. ribosomes, dna/rna polymerase)
smal proteins enter the nucles through diffusion
Why else is active transport more desirable than diffusion in terms of nuclear import/export ? wouldnt want large molecule heading freely to nucleus. don't want any type of rna's coming out of nucles that are properly spliced, only want mature rnas coming
NLS (import of proteins to nucleus)
Proteins that contain a nuclear localization signal (NLS) localize to the nucleus (start in cytosol and move to NUCLEUS)
are lysine-arginine rich
question: if you were studying nuclear imports & see seq with lys-arg what would u do to prove that your prot
Directionality of nuclear import
Ran-GAP stimulates GTP hydrolysis in the cytosol and Ran-GEF restores Ran-GTP in the nucleus
Ran-GAP is localized to the cytosol
NES (Export of proteins to cytoplasm
Nuclear export requires a nuclear export signal (NES) and receptor (similar mechanism to nuclear import)
Ran
Ran regulates the binding and/or release of cargo from the export/import receptor
GTP hydrolysis
RanGAP hydrolyzes GTP, releasing receptor and cargo
the binding of Ran-GTP might cause nuclear import receptors to release their cargo
Binding of import receptor by Ran-GTP alters NLS recognition site - forcing release of cargo
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