Membranes divide the cytoplasm of eukaryotic cells into WHAT 2 ITEMS
distinct compartments and interconnected networks of canals
T OR F?
Membrane-bound structures of various diameter containing material of different electron density are found in all eukaryotic cells
TRUE
DEFINE Endomembrane system
ANYTHING THAT HAS TO DO WITH THE INSIDE OF THE CELL AND THE MEMBRANE.
Biosynthetic pathways
Secretory pathways
Endocytic pathways move materials into cells
Sorting signals
DEFINE EXOCYTOSIS/ENDOCYTOSIS
EXOCYTOSIS...OUT OF CELL..OUT CYTO
ENDOCYTOSIS..INTO CELL...IN CYTO
1974 Nobel Prize for 3 cell biologists: Study of Cytomembranes
De Duve (University of Louvain in Belgium),
Claude &
Palade (both of Rockefeller University)
James Jamieson & George Palade - worked with WHAT?
pancreas acinar cells; followed secretory protein from synthesis to secretion & determined individual steps even though all of them occurred simultaneously.
PULSE/CHASE METHOD
__________ ________cells have a particularly extensive endomembrane system; ideal for study by autoradiography
Pancreas acinar
METHOD KNOWN AS PULSE/CHASE METHOD.
WHAT PROCESS ALLOWS ONE TO OBSERVE BIOCHEMICAL PROCESS?
Autoradiogram...PULSE/CHASE
NAME A HOT/COLD AMINO ACID.
HOT....SULFER35..PULSE..DISULFIDE BRIDGES
COLD...REGULAR SULFER
WHAT IS THE 1ST STEP IN AUTORADIOGRAPHY?
Incubate tissue with hot amino acids briefly —> incorporated into digestive enzymes as they are made on ribosomes...S35 GOES FOR DISULFIDE BRIDGES.
IN PALADE & JAMIESON'S PULSE/CHASE METHOD, WHAT ARE THE HOT(PULSE) & COLD (CHASE)?
AMINO ACIDS
T OR F ?
IN AUDIORADIOGRAPHY....The longer the chase, the farther the hot (radioactive) proteins made during the pulse will have traveled from their synthesis site (the RER) within the cell
TRUE
BECAUSE, unlabeled amino acids (chase), which lasts for varying time periods, protein synthesis continues using nonradioactive amino acids
THE SECRETORY PATHWAY IS ALSO KNOW AS WHAT?
BIOSYNTHETIC PATHWAY
HOW WOULD YOU USE A VIRUS AND GFP?
vesicular stomatitis virus gene (VSVG) is fused to GFP gene; viruses useful since they turn cells into factory for producing one or a few viral proteins.
Cell begins to make massive amounts of VSVG protein in RER
DEFINE temperature-sensitive mutants
Mutants of this type that function normally at reduced (permissive) temperature, but not at elevated (restrictive).
CONTROL TEMP, CONTROL MOVEMENT.
DOESN'T GO IN REVERSE.
THE vesicular stomatitis virus gene (VSVG) REACTIONS TO TEMPERATURE: 40C & 32C
40C: GROWS, BUT DOESNT LEAVE ER
32C: LEAVES ER & GOES TO GOLGI AND SO ON.
VSVG IS KNOWN AS temperature-sensitive mutant.
Cell homogenization & organelle isolation (CELL FRACTIONAL) techniques were pioneered by WHO & WHEN?
Claude & De Duve (1950s & 1960s)
Purification of subcellular fractions by density gradient equalibrium centrifugation. (SUCROSE) LYSOSMES, MITOCHONDRIA & PEROXISOMES.
WHERE DOES EACH FALL?
65,000g @ 2HRS
Vesicles from different parts of Golgi were found to have enzymes that add different sugars to the ends of growing ____ chains of ________ & _________.
CHO
glycoprotein or glycolipids
EXAMPLE OF glycolipids
SUGARS THAT MAKE ANTIGENS OF RBC
Vesicles from different parts of Golgi were found to have enzymes that add different sugars to the ends of growing CHO chains of glycoprotein or glycolipids.
HOW CAN ONE USE THIS INFO IN ORDER TO USE GOLD AS A TAG TO STUDY GOLGI?
Purify these enzymes from the microsomal fraction; use them as antigens to make antibodies & attach gold particles to the antibodies, locations of which in Golgi membranes can be seen in EM.
Revealed role of Golgi complex in stepwise assembly of complex carbohydrates
studied properties of rough microsomal fraction (cell-free systems, isolated parts of cell)...WHO & WHEN?
Palade & Siekevitz, et al. (Rockefeller University, 1960s)
DEFINE Cell-free liposomes
vesicles whose walls consist of an artificial bilayer created from purified phospholipids, used to study specific roles of proteins involved in budding
T OR F?
Mammalian (EUKARYOTIC) cell-free systems can use yeast proteins to facilitate vesicle transport.
TRUE.
can "cure" yeast biosynthetic pathway mutants by genetically engineering them to carry normal mammalian genes
Membranes of the ER enclose a ______ space (cisternae)
lumenal
An interconnecting network of tubular membranes elements
Smooth ER (SER)
Functions include:
Synthesis of steroids in endocrine cells gonads and adenal cortex
Detoxification of organic compounds in liver via oxygenases: occasional faulty carcinogen conversion
WHAT THE HELL IS IT?
SMOOTH ER (SER)
Sequestration of Ca+ in sarcoplamic reticulum (MUSCLE)... WHAT ORGANELLE OF THE CELL DOES THIS?
SMOOTH ER (SER)
___________ oxydizes thousands of hydrophobic compounds- convert them to more hydrophylic/excretable
Cytochome p450
Proteins synthesized on ribosomes of RER
Secretory,
integral membrane,
proteins of organelles
Proteins synthesized on “free” ribosomes.
Cytosolic proteins,
peripheral membrane proteins,
nuclear proteins
chloroplast and mitochondrial proteins
WHAT SIDE ARE THE RIBSOMES ON IN THE RER?
ribosomes on the cytosolic side of continuous flattened sacs (cisternae).
FACES OUT INTO THE CELL, NOT TOWARDS NECLEUS.
WHERE IN THE FUCK DO YOU FIND MUCOPROTEINS?
Mucous secreting goblet cell from a rat colon. ER products move to golgi and are packaged in membrane as concentrated mucoproteins.
5 STEPS TO SIGNAL SEQUENCE ON MEMBRANE BOUND RIBOSOME ON RER.
1. Signal sequence binds a signal recognition particle (SRP)
2. Then attaches to an SRP receptor on the ER membrane 3. SRP and SRP receptor must both bind to GTP in order to interact (G proteins)
4. Sequence is being moved through membrane as it is being synthesized i.e. a co-translational event
5. Protein conducting channel is blocked by chaperone protein on lumenal side of the ER
HOW DO YOU MAKE NON-MEMBRANE ASSOCIATED PROTEIN. (secretory protein, lysosomal enzyme, hormone)
In the Model of synthesis of secretory protein (or lysosomal enzyme) on membrane-bound ribosome of RER, name the chaperone.
BiP
WHAT THE HELL IS A TRANSLOCON CHANEL?
ITS IN THE ER MEMBRANE
synthesis of secretory protein...2 TYPES OF RNA ARE INVOLVED. WHAT/WHERE ARE THEY?
Messenger RNA binds to free ribosomes in the cytosol.
DEFINE nascent protein
A protein as it is being formed by a ribosome before it folds into its active shape.
synthesis of integral membrane protein..NAME THE 6 STEPS
synthesis of integral membrane protein.. THE STOP TRANSFER SEQUENCE...HYDROPHOBIC OR HYDROPHILLIC....WHY?
HYDROPHOBIC.
IT'S INTERGRAL, GOING THRU BILAYER. CANT BE HYDROPHILLIC OR IT WOULD WANT TO LEAVE THE CELL.
WHAT IS THE PURPOSE OF A CHAPERON IN THE SYNTHESIS OF INTEGRAL MEMBRANE PROTEIN?
chaperone protein must block the open channel (TRANSLOCON) on the luminal side and act as a permeability barrier.
T OR F ?
Maintenance of membrane asymmetry...This orientation doesn’t change as it moves from ER-GC-PM.
TRUE.
TO MAINTAIN THE MEMBRANE ASYMMETRY WHEN A MEMBRANE IS PRODUCED AND CARBS ADDED ON PROVIDE INFO ON SIDENESS OF PROTEINS, WHAT SIDE?
cisternal (OUTSIDE) side of vesicle material.This becomes the exoplasmic side for plasma membrane destined proteins post fusion.
__________ transfer proteins that move lipids between membranes
Phospholipid
Lipids are inserted into the outer leaflet of ER and are modified by WHAT? NAME 4
FLIPASE
Conversion of one type phospholipid to another
Selective inclusion in new vesicles
Phospholipid transfer proteins that move lipids between membranes
T OR F?
Modifying the lipid composition of membranes AS THE MEMBRANE TRAVELS THRU ER GC PM EITHER START HIGH AND GO LOW OR START LOW AND GO HIGH.
TRUE
This is site of synthesis for proteins destined for secretion, lysosomes, or membranes.
The endoplasmic reticulum (ER)
The site of lipid and steroid synthesis. New membrane is made here.
smooth er
DEFINE CISTERNAL SIDE.
THE OUTER SIDE. LUMEN IS INSIDE.
INTEGRAL PROTEIN SYMETRY
LIPIDS
HORMONES
WHERE ARE THEY PRODUCED?
SMOOTH ER
NAME 3 PHOSPHOLIPIDS
PHOSPHATIDYLCHORLINE
PHOSPHATIDYLERSINE
SPHINGOMYELIN
Three mechanisms for changing phospholipids (PL) between ER-GC-PM...NAME THEM
1) head groups are modified enzymatically
2) Membrane forming vesicle contains different PL composition than what it buds from
3) Pl can be removed from one membrane and inserted into another via PL-transfer proteins.
WHAT THE HELL IS THIS?
Three mechanisms for changing phospholipids (PL) between ER-GC-PM.
1) head groups are modified enzymatically
2) Membrane forming vesicle contains different PL composition than what it buds from
3) Pl can be removed from one membrane and inserted into another via PL-transfer proteins
_________ add carbohydrates to almost all proteins produced by membrane bound ribosomes (glycoproteins)
Glycosyltransferases
WHERE DOES Glycosylation OCCUR? WHAT IS IT?
OCCURS IN THE RER. ITS WHEN YOU ADD CARBOHYDRATES TO PROTEINS PRODUCED MY MEMBRANE BOUND RIBSOMES (GLYCOPROTEINS)
DURING Glycosylation in the RER, Glycosyltransferases add carbohydrates to almost all proteins produced by membrane bound ribosomes (glycoproteins). Sugars are attached in a ordered sequence.The core carbohydrate chain is assembled on a lipid carrier. WHAT IS THE CARRIER?
Dolichol phosphate
Dolichol phosphate...WHERE IS IT FOUND AND WHATS IT FUNCTION?
Glycosylation in the RER. IT IS A LIPID CARRIER THE THE CORE CARBOHYDRATE CHAIN IS ASSEMBLED UPON.
DURING Glycosylation in the RER, the core carbohydrate chain is assembled on a lipid carrier, Dolichol phosphate.
WHAT HAPPENS NEXT?
The core carbohydrate is transferred to the polypeptide by oligosaccharyltransferase. It is modified en route to the Golgi complex.
(OLIGO MEANS SHORT.)
SUGARS DON'T LIKE MEMBRANE WHY?
THEY CONATIN ALOT OF ALCOHOL GROUPS ON THEM.
Steps in synthesis of core portion of N-linked oligosaccharides in RER.
After Glycosylation in the RER what are the 2 steps to vesicular transport protein/sugar product from the lumen of the RER to the GC?
Membranes and luminal proteins move to the tips of the RER.
Transitional elements (ie, RER tips) form the first transport vesicles.
THEN OFF TO THE GC
Cytoplasmic membrane systems are characteristic of what types of cells?
A. prokaryotic cells
B. eukaryotic cells
C. bacterial cells
D. all cells
eukaryotic cells
Pancreatic cells that produce and release digestive enzymes engage in what type of secretion?
A. regulated secretion
B. constitutive secretion
C. transport secretion
D. none of the above
regulated secretion
Which of the following cell types would be the best model system for studying secretory granules?
A. muscle cells
B. yeast cells
C. epithelial cells
D. pancreatic cells
pancreatic cells
You are interested in studying the asymmetric (coming from just one cell side) release of digestive enzymes from epithelial cells that line the digestive tract. Which of the following techniques would be best for these studies?
A. cell fractionation
B. homogenization
C. autoradiography
D. all of the above
autoradiography
why microsomes can't be seen in cells viewed with the electron microscope?
They are artifacts of homogenization and centrifugation.
**THIS IS FROM THE ONLINE QUIZ AND YES THIS WAS THE CORRECT ANSWER**
Which of the following proteins would not be found in the smooth endoplasmic reticulum?
A. Ca2+-pumping enzymes
B. cytochrome P450
C. glucose 6-phosphatase
D. signal peptidase
signal peptidase
Which of the following groups of proteins probably lack a signal sequence?
A. acid hydrolase enzymes synthesized in macrophage cells B. glycolytic enzymes synthesized in liver cells
C. polypeptide hormones synthesized in endocrine cells
D. antibody hormones synthesized in plasma cells
glycolytic enzymes synthesized in liver cells
Which of the following is not an essential component of the complex that directs a nascent protein into the lumen of the RER?
A. protein disulfide isomerase
B. SRP
C. SRP receptor
D. GTP-binding protein
SRP
*****THIS IS FROM THE ONLINE QUIZ AND YESTHIS WAS THE ANSWER*******
WHEN A PROTEIN, INTEGRAL OR SECRETORY, GETS MISFOLDED WHERE IS IT DESTROYED?
CYTOSOL. UBIQUEIN ATTACHES AT LYSOSOMES ATTACK IT.
If you compared the proteins in a cis Golgi compartment with those in a trans Golgi compartment, you would find WHAT?
the proteins in the cis compartment are glycosylated, whereas those in the trans compartment are glycosylated and contain modified amino acids
You are tracing the path of a secretory protein from its synthesis to its export from a cell. RADIOACTIVE TAGED. List the order in which the proteins of these fractions first exhibit radioactivity.
I secretory vesicles
II Golgi complex
III rough ER
IV smooth ER
V nucleus
III--->II---->I---->out of the cell
Of the following processes that occur during protein trafficking, which involve GTP?
A. protein translocation across the ER membrane
B. disassembly of the COP coat on a transport vesicle
C. fusion of non-clathrin coated vesicles with target membrane
D. all of the above
ALL THE ABOVE
You have labeled the lipids on a patch of rough ER membrane with a fluorescent probe. After a few minutes, the probe shows up in the membranes of the cis Golgi. Now you treat the cells with the drug brefeldin A. Where might the fluorescent probe show up next?
A. trans Golgi network
B. endoplasmic reticulum
C. plasma membrane
D. secretory vesicles
endoplasmic reticulum
ANOTHER ONE FROM THE ONELINE TEST
v-SNARE proteins are to transport vesicles as KDEL sequences are to ? .
A. t-SNARE proteins
B. escaped ER proteins
C. ERGIC vesicles
D. coat proteins
t-SNARE proteins
Which type of vesicle of the trans Golgi network would be most likely to carry hormones destined for regulated secretion?
A. lysosomal vesicles
B. clathrin-coated vesicles
C. non-clathrin-coated vesicles
D. all of the above
clathrin-coated vesicles
COPII-coated vesicles are to anterograde movement as ______ are to retrograde movement.
COPI COATED VESICLES
Of the following, which would be least likely to be found in lysosomes?
A. acid hydrolase enzymes
B. a half-digested mitochondrion
C. nucleic acids
D. a high concentration of protons
nucleic acids
Uptake of low-density lipoproteins (LDL) occurs by _____, whereas retrieval of plasma membrane after extensive secretory activity occurs by ________.
