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Cell Bio Exam 2: Protein Sorting
Terms in this set (66)
What is ER structure?
Network of membrane: enclosed tubules and sacs that extends from the nuclear membrane throughout the cytoplasm; Hard to isolate and keep intact; micrososmes=isolated after breaking into cell
What is the Rough ER?
covered by ribosomes and associated with protein synthesis
What is the Smooth ER?
ER that has no ribosomes and involved with lipids
What is the Transitional ER?
where vesicles exit to the Golgi apparatus and carry lipids and proteins
What is the secretory pathway?
Rough ER -> Golgi -> secretory vesicles -> cell exterior
Secretion: release of protein from cell in extracellular space
How did Palade's Experiment define the secretory pathway?
studied pancreateic acinar cells that secrete digestive enzymes into small intestine; newly synthesized proteins labeled with radioisotopes(''pules'')
What is the pulse-chase experiment?
incubate organ slices in medium containing Leucine for 3 min
incubate organ slices in medium containing Leucine for varying periods up to 117 minutes
Goes from ER to Golgi and then secreted into vesicles and goes to cell exterior
What is a signal sequence?
The sequence within a protein that directs the protein to a particular organelle.
What does the signal recognition particle (SRP) do?
binds to the signal peptide and brings the signal peptide and its ribosome to the ER
What does a SRP receptor do?
allows N-terminus attached to SRP and protein to bind when GTP is present
What is a Sec 61 translocon?
a translocon through which proteins translocate
What is signal peptidase and what does it do?
Enzyme that removes a terminal signal sequence from a protein once the sorting process is complete.
What is post-translational targeting?
protein synthesized completely, ribosome makes its way to ER and has to have Sec 60
What is co-transitional targeting?
while protein is translated, ribosomes makes itself to surface of ER
How are proteins inserted into ER?
• Stop transfer sequences in a protein cause translocation to stop
o They are associated with stretches of hydrophobic amino acids which integrate into the membrane
• Protein will be integrated in plasma membrane
• N terminus ends up on outside of cell
• Integral membrane spanning proteins first associate with the membrane as they translocate into the ER
o Multiple signal sequence can expand the membrane multiple times
To get protein back into the translocon again
o Stop transfer sequences- arrest translocation and enable membrane spanning to occur
o sequences not cleaved can enable the C terminus to cross into the ER lumen
What's the ER's role in protein folding?
Chaperone proteins and PDI help facilitate correct folding of proteins in the ER
What's the ER's role in disulfide bond formation?
protein disulfide isomerase breaks and reforms disulfide bonds
What's the ER's role in N-linked glycosylation?
attachment of carbohydrate to nitrogen atom of asparagine side chain
• Proteins are N-glycosylated within the ER lumen
• The amino acid asparagine is glycosylated within the consensus sequence of Asn-X-Ser/Thr
• Core glycosylation occurs in the lumen of the ER (has to have some sort of leader sequence)
• Implies that the glucoses get trimmed off which can be apart of the folding quality control
• End up with high mannose structure
• All on outer surface of cell or secreted
• Has to have internal leader sequence (allows C terminus to get inside and N terminus out), stop transfer sequence (gets into membrane), Asn-X-Ser/Thr to end up on plasma membrane.
• Ex: If there is just a leader sequence and no other sequences it would be secreted out of the cell
What's the ER's role in the addition of GPI anchors?
• GPI anchors are attached within the ER lumen
• The proteins start with a membrane spanning C-terminus which is cleaved upon GPI addition
• Start out as transmembrane proteins and ends up being membrane associated
• If goes to plasma membrane= ends up external because requires GPI anchor in lumen of ER
• C-terminal transmembrane stretch to require GPI anchor
• GPI proteins become concentrated in lipid rafts
What's the ER's role in quality control?
• Calreticulin help mediate the folding of glycosylated proteins
• Channel the protein which requires it to fold= glucose removed
• Misfolded proteins transported out of ER where they are degraded
What is ERAD?
Endoplasmic-reticulum associated process where misfolded proteins are sensed and degraded
What is UPR?
unfolded protein response and where unfolded proteins are accumulated and degraded
o If overwhelming degradation, cell death happens.
What is the functional role of ER in synthesis of membrane lipids?
phospholipid synthesis occurs primarily on the cytosolic face of the ER; flipase activity associated with ER to generate a balance of phospholipid concentration
newly synthesized lipids added only to cytosolic face half of bilayer (PI, PS, PE)
on outside of bilayer: PC, SM, glycolipids, GPI's
Where do proteins exit ER?
• Proteins and phospholipid molecules are exported from the ER in vesicles that bud from the transitional ER, move through the ER-Golgi intermediate compartment, and then to the Golgi apparatus
o ER-Golgi intermediate compartment: some protein folding can be completed, disulfide bond formation, concentration of proteins, it is a stop from ER to Golgi
o ER export signal: for proteins to know how to get into vesicle and leave ER
"vesicle targeting signals"
• Retrieval of resident ER proteins (often via C-term KDEL sequences)
o C-terminus of protein attaches to KDEL sequence
Sorted, headed to Golgi and returned to ER
• KDEL receptor in membrane which allows protein to bind to receptor
o KDEL receptor dissociates when sorted into vesicle
pH is slightly different which allows protein to detach from receptor
o Three proteins that have KDEL sequence: PDI (protein that lives in ER), chaperones (BIP), flipases, sec60-1 translocon, signal peptidase, basically anything that involves translocation into the ER
What is the cis Golgi network?
• What accepts vesicles from the ER and proteins
• Receives molecules from the ERGIC with some modifications beginning here
• Commonly oriented towards nucleus
• Cis becomes medial and medial becomes the trans
What is the trans Golgi network?
• The sorting and distribution center as proteins bud off and head to final destination
• Oriented towards plasma membrane
What is ERGIC?
ER-Golgi intermediate compartment: some protein can be completed disulfide bond formation, concentration of proteins, it is a stop from ER to Golgi
How are secretory proteins transported through the Golgi?
• Cisternal maturation, stable cisternae model
o evidence for each of these processes include electron micrographs and the disappearance of the Golgi as an organized structure if vesicle transport from the ER is blocked
• Regulated secretion: insulin response, neurotransmitters
What is protein glycosylation?
Asn-X-Ser/Thr gets glycosylated
How are lysosomal proteins targeted to lysosome?
1. targeting to the lysosome by phosphorylation of mannose-6-phosphate—a mannose-6-phosphate receptor in the trans Golgi network transports them to endosomes and on to lysosomes
2. Single pass lysosomal transmembrane protein:
a. N-terminus leader, stop transfer/hydrophobic stretch, Asn-X-Ser/Thr, Vesicle targeting sequence, Mannose-6-phosphate
How does a vesicle coat form and what's its function?
o Two GTP binding proteins promote coat assembly
o Arf for COP I and clathrin coats
o Sar for Cop II coats
o Creates a scaffold around where vesicle buds
o To form budding with ArfGDP and attach with Golgi, need to activate Arf by GDP into GTP by replacing molecule with entirely new GTP, Arf would be bumped to guanine nucleotide exchange factor (GNEF trans-face Golgi) and is activated. GTP hydrolysis on Arf would lead to coat disassembly
What do COPI-coated vesicles do?
coated vesicles bud from the ERGIC or Golgi and carry their cargo backwards, returning proteins to earlier compartments
What do CopII vesicles do?
coated vesicles carry secretory proteins from the ER to the ERGIC and on to the Golgi
• COP II vesicle budding:
o Sec 12p: GNEF and activates Sar
o Sec 23 and 24: adaptor proteins
o Sec 13 and 31: Cop II coating proteins
o If unable to bud from ER--no Golgi and large ER
What happens in the assembly of vesicle coats?
To form budding with Arf-GDP and attach with Golgi, need to activate Arf by GDP into GTP by replacing molecule with entirely new GTP, Arf would be bumped into GNEF and is activated.
GTP hydrolysis on Arf would lead to coat disassembly
What is Clathrin and what does it do?
coated vesicles transport in both directions between the trans Golgi network, endosomes, lysosomes, and plasma membrane
How do transport vesicles recognize their correct targets (Rab proteins)?
• The Rab family of small GTP-binding proteins plays a key role in accurate vesicle targeting
• Rab proteins mark different organelles and transport vesicles, so their localizations on the correct membrane is key to establishing the specificity of vesicular transport
• Rab proteins on vesicles, recognize and bind to tethering proteins allowing binding to correct target membranes
• More than 60 different Rab proteins in the cell
o Rab tethering protein specificity that allows Rab to end up in specific location
• Tight junction limits protein diffusion
• A rab protein is only activated when presented to the correct Rab-GEF
• This ensures that it is functional only at the location where it should be
What are v-SNAREs and t-SNAREs involved in?
• Vesicle fusion is mediated by interactions between pairs of transmembrane proteins (called SNAREs) on vesicle and target membranes (v-SNAREs and t-SNAREs)
• v-SNARE—t-SNARE binding and conformational changes provide the energy to bring the two bilayers close enough to destabilize them and fuse
• t-SNARE: target membrane
• v-SNARE: on vesicle
o Both facilitate fusion and accurate targeting based upon which v-snares bind to which t-snares
• Conformation change facilitates fusion between t-snares and v-snares
What is a lysosome and it's function?
digestive organelle of the cell that uses enzymes to break down biological polymers
• Contains about 50 different degradative enzymes
• mutations in genes that encode these enzymes result in lysosomal storage diseases
• lysosomal environment: pH 5
• cytoplasm: pH 7
o Uses direct active transport b/c ATP dependent for protons to enter the cell
In other words: To maintain the acidic pH, a proton pump in the lysosomal membrane actively transports protons into the lysosome
How do lysosomes form?
• Differential Centrifugation:
o Series of spins of increasing force where supernatant of one spin is used in the next spin and so on.
800 G= force of gravity on Earth
• Lysosomes were discovered by analyzing subcellular fractions prepared by differential centrifugation
• Acid phosphatase, a lysosomal enzyme; cytochrome oxidase, a mitochondrial enzyme; glucose-6-phosphaste, an ER enzyme served as marker enzymes
o Phagocytosis: a pseudopod reaches out and will engulf a bacteria or dead cell (macrophage)
a macrophage knows a cell is dead and needs to be rid of because the cell breaks open and inner membrane is exposed which allows the macrophage to recognize it
to degrade internal organelles that have been damaged
What is Endocytosis?
o Using vesicles to bring things into the cell
o endosome matures into late endosome which becomes a lysosome or could fuse with a previously existing lysosome
What is Phagocytosis?
•bacteria, dead cell, virus, ect. ; specialized cells such as macrophages take up and degrade large particles, including bacteria, cell debris and aged cells
o The particles are taken up in phagocytic vacuoles (phagosomes), which fuse with lysosomes to become phagolysosomes and be digested
What is Pinocytosis?
(cell drinking) is a property of all eukaryotic cells
What is receptor-mediated endocytosis?
• a mechanism for selective uptake of specific macromolecules
o Macromolecules bind to specific cell surface receptors concentrated in specialized regions called clathrin-coated pits
o The pits bud from the membrane with the help of dynamin, to form small clathrin-coated vesicles; these then fuse with early endosomes
o Dynamin is a protein that helps produce clathrin-coated vesicle and bud
It knows to go to the early endosome b/c it's marked with a rab protein and specific tethering protein at the membrane of the endosome
• Ex: how you take cholesterol out of your blood
What is the uptake by caveolae involved in endocytosis?
o Caveolae are small invaginations of the plasma membrane organized by a protein called caveolin
o Caveolae carry out receptor-mediated endocytosis via specific transmembrane receptors
What is the advantage of having a nucleus?
compex regulation of gene expression
What is structure of the nuclear envelope?
double membrane surrounding the nucleus
What is the nuclear lamina?
proteins network that lies the inner membrane of the nucleus
made of cross-linked lamin polymers
to assemble: dimers polymerize which gives the structural integrity of the nucleus
What causes the nucleus to disappear?
degradation of the lamina by phosphorylation which allows proteins to repel each other by having the same charge
What do nuclear pores do?
allow material to move in and out of the nucleus
proteins and RNA molecules and transported through energy-dependent transport
What is nuclear localization signal/nuclear export signal?
signal present on protein that causes it to go through the channel to end up in the nucleus
What is karyopherin?
AKA importins and exportins. Transport molecules that facilitate movement of other molecules through the NPC.
What is importin?
imports cargo into the nucleus (has to have nuclear localization signal and bound by it to go into nucleus)
What is exportin?
A transporter receptor recognized by the nuclear pore complex.
Ran is critical to protein export in nucleus
What is Ran?
efficiently redistributes proteins back into cytosol to bring proteins with nuclear localization signal
What is Ran GEF?
resides in nucleus, directly implicated in export, implicated in import because it recycles importin by GTP
What is Ran GAP?
activating protein that resides in the cytosol
How are RNAs transported out of the nucleus?
ribosome is disassembled in the nucleus, RNAs are transported as RNPs
What's the basic structure of the nucleolus and its fucntion?
blocks light because there are so many molecules present due to no membrane
rRNA is ranscribed here and used for assembling ribososmes
18S, 5.8S, 28S are transcribed on one transcript and are cut into those three pieces
What's the structure and function of the mitochondria?
consists of outer membrane, inner membrane space (oxidative phosphorylation). inner membrane (cristae), matrix center
What is the mitochondrial genome?
circular DNA molecules present in multiple copies. Encode only a few proteins that are essential for oxidative phosphorylation. Encodes all rRNA
What is evidence for the endosymbiotic origin of mitochondria?
From prokaryotic organisms. Mitochondrial genome
What are some of the genes present in the human mitochondrial genome?
13 proteins involved in electron transport and oxidative phosphorylation
majority of proteins are encoded by nuclear genome, made in cytosol and need to be imported
What is Tim?
translocon if inner membrane
What is Tom?
translocon of outer membrane
What happens to proteins when imported into mitochondria?
targeted by the matrix amino-terminal sequences that are removed by proteolytic cleavage after import
How does protein get inserted into second membrane of mitochondria?
Hsp70 binds and conformational change happens and inner membrane has to be polarized
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