Unit 2 Flashcards
How many proteins are found in eukaryotic cells
~10,000
Where does protein synthesis begin
Free ribosomes in ER
What directs the ribosome to sit down on the ER
Signal sequence at the N terminal end of the protein. As soon as the signal sequence of AAs exits the ribosome translations stops and ribosome sits dow on ER.
General pathway of a secretory protein
cytosol (free ribosome)
Bound to ER
Moved through translocon into ER
Modified in ER
Moved by budding into Golgi
Modified in Golgi
Packed in vesicle and moved out
What does this picture show
ER is mostly rough
SER is in the middle
Lumen of ER is continuous
Homegenization of cells causes
ER to break up into microsomes. Rough and smooth separate
What fraction of density gradient centrifugation contains secretory proteins
Rough microsomes
Why are secretory proteins in the ER smaller (lower mol. wgt) than those not yet in ER or unable to enter ER?
Signal sequence is only cleaved off in ER
If you add microsomes AFTER protein is completed then they can’t enter.
How can you tell that a protein has been extruded into a microsome?
Resistant to proteases unless treated with a detergent.
Become glycoslyated by enzymes only found within microsomes.
How does the signal sequence direct a protein to the ER?
A signal sequence binds a signal recognition particle.
SRP stops translation.
Moves to ribosome where SRP binds to the SRP receptor.
SRP is a
Riboprotein complex
Translocons
Open up once SRP binds receptor.
Do not require ATP (ATP used in translation operates the translocon)
Open only to protein and not other small molecules.
How does Post-Translation Translocation differ from production of a typical secretory protein
Riosome does not attach to ER.
Protein is completed in cytosol.
Signal sequence moves protein to translocon.
Binds / uses ATP to prevent slipping backwards in translocon.
What is the first part of this picture showing
Proteins can have different orientations in the membrane (N or C terminus in the cytosol or lumen)
How is orientation of multi-pass proteins determined
Even numer of passes = N and C term on same side.
Odd number of passes = N and C term on different sides.
**Proteins will not function without proper orientation in membrane.
Type 1 Membrane proteins
C term in cytosol
Majority of protein in lumen
Type II protein
N term in cytosol
Majority of protein in lumen
Type III Protein
C term and majority of protein in cytosol
**Tail anchored = C term embedded in membrane
Type IV Protein
Multipass proteins
Type IV - A: N and C on same side
Type IV-B: N and C on different sides
True or False: proteins embeded in the RER remain in the membrane as they move to their final destination.
Which end is considered to be the “tail” of the protein?
C terminus
Topology of a protein
Membrane spanning segments are usually made of
20 - 25 hydrophobic amino acids
Type II Proteins
Do NOT have a cleavable ER signal sequence
Oriented wth hydrophilic N-terminal region on cytosolic face
Oriented with hydrophilic C-terminal region on exoplasmic face
Type III
Same orientation as Type I due to hydrophobic membrane-spanning segment at N - terminus.
DO NOT contain a cleavable signal sequence
Tail Anchored Proteins
hydrophobic segment at C-term that spans membrane
What are the three main types of topogenic sequences used to direct proteins to the ER membrane?
N-terminal signal sequences
Stop transfer signal sequences (internal)
Signal-anchor sequences (internal)
What do stop-transfer anchor sequences do?
Stop passage of polypeptide chain through the translocon
Anchor the polypeptide to the membrane
Both type II And type III have these
What is the key difference between type II and III proteins?
orientation of hydrophobic transmemrane segment as it binds to the hydrophobic signal-sequence inding site at the edige of the Sec61alpha
What determines the orientation of a signal anchor sequence in the membrane
high density of positively charged amino acids adjacent to one end of the hydrophobic segment
Type II have + residues on N term side
Type III have + residues on C term side
**Mutations can cause these to flip
Why don’t tail anchored proteins get treated the same?
hydrophobic region at C terminus is only “visible” once the protein is done being translated and has left the ribosome.
Do not use SRP / SRP receptor
Use Get3 pathway and GTP hydrolysis
What serve as signal anchors in multipass proteis
First N-term alpha helix
odd numbered sequences
**Even numbered act as stop-transfer anchor sequences
What is the difference between a signal anchor and a stop transfer sequence?
Signal anchor: oriented with N - term toward cytoplasm
Stop - transfer: N - term toward exoplasmic face
What are the 4 prinicple modifications of proteins BEFORE they reach their destination
- Covalent addition and processing of carbs
- Formation of disulfide bonds
- Proper folding
- Specific proteolytic cleavages
WHat is the structure of all N-linked oligosaccharides
three glucose
nine mannose
two N-acetylglucosamine
How are N-linked oligosaccharides modified
addition or removal of monosaccharides in ER or Golgi
A core of 5 - 14 residues is conserved
Where are the enzymes that add monosaccharides to N-linked oligosaccharides found
on the cytosolic or luminal faces of the ER membrane
What are the steps to create a final N-linked Oligosaccharide
glycosidases remove 3 glucoses and one mannose
Three glucoses are a signal that the side chain is ready to be added to a protein
What is the purpose of adding N-linked oligosaccharides?
Promote proper folding of proteins
increase stability
Cell to cell adhesion (if on cell surface)
Induce immune response
Where do disulfide bonds form?
in the lumen of the rough ER
in solube secretory proteins
on exoplasmic domains of membrane proteins
What order to disulfide bonds form in?
first: stabilize small domains
Second: stabilize distant sections
What is the enzyme that creates and changes disulfide bonds
protein disulfide isomerase
What is contained in lysosome
low pH
acid hydrolases
Only active at low pH so this protects cell
What plant organelle can function like a lysosome
vacuoe
What directs proteins to lysosome
A signal sequence:
a phosphorylated mannose residue
“signal patch” found in AA chain
Why are lysosomal disorders called storage diseases
missing hydrolytic enzyme so something doesn’t get broken down and it will be stored in lysosome.
What side of golgi has vesicle budding
trans
Vesicles budding from trans gogli can contain what
proteins to be added to cell membrane
proteins for other organelles in the cell
proteins / materials for secretion (hormones, enzymes, neurotransmitters etc)
Transport into the cell via a vesicle includes what
endocytosis
(pino / phago)
recepter mediated endocytosis
Phagocytosis
Not random.
Specific
Pinocytosis
Random and nonspecific
Receptor mediated endocytosis
receptors randomly caught up in pits
receptors bind molecules and then they are also in pit.
molecules released during pH change in late endosome
Fate of receptors in receptor mediated endocytosis
recycled
degraded
transcytosis: moved to a different domain of membrane
What two pathways use clathrin coated vesicles
trans golgi to lysosome
receptor mediated endocytosis
What are the three types of proteins that coat vesicles
clathrin
cop1
cop2
COP = coatomer
What is the process that forms / buds / pinches vesicle
- surface proteins spontaneously assemble on membrane
- adaptin binds the proteins to the membrane by binding its receptor
- protein pulls on membrane and creates the budding of vesicle
- Dynamin acts like a spring and squeezes off the esicle
GTPase examples
Sar 1
ARF
GTPase activation
Inactive with GDP
Active with GTP
V class snares
are attached to donor organelle vesicle
T class snare
are attached to fusing organelle
How do Rabs and snares help with vesicle fusion?
Rab GTPases help vesicle find the correct membrane and forma weak interaction.
V snares then bind with T snares and pull the vesicle into the membrane
What are the key areas in which cytoskeleton plays a role?
Shape changes
Coordinated movement (directional)
Division
Organize intracellular space
What are the three types of cytoskeleton?
Micofilaments (actin)
Intermediate filaments
Microtubules
Where will actin be in each of the following cell types:
polarized cells
motile cells
Dividing cells
Polarized: in the core structure
Motile: leading edge
Dividing: Contractile ring
Shape of each of the following:
Actin (microfilaments)
Intermediate filaments
Microtubules
Actin: helical
Intermediate: rope like
Microtubule: hollow, cylindrical
Which type of cytoplasm is rigid
Microtubules (still dynamic)
G-actin is added to which end of the growing actin polymer?
+ end.
In vitro - end will grow, but in vivo it doesn’t grow quickly.
What are the three steps in actin polymerization
- Nucleation **Rate limiting step
- Elongation at + end
- Steady state where rate of addition = rate of loss
What is Cc?
G - actin critical concentration.
IF [Gactin] > Cc G actin will polymerize into F actin until Cc is reached.
IF [G-actin] < Cc, F actin will depolymerize into G-actin until Cc is reached.
G actin monomers are able to bind _____________so are classified as ________________.
ATP
ATP binding proteins
What is the role that ATP plays in actin polymerization?
What causes “treadmilling” in actin polymerization?
G actin added at + end as G actin is lost at - end.
What protein plays a role in acin polymerization
Actin binding protein
Three toxins: cytochalasin, lactrunculin, and phalloidin interfer with actin what is the mechanism of each?
cytochalasin: causes depolymerization
latrunculin: causes depolymerization
phalloidin: causes stabiliation
How does cofilin aid in depolymerization of actin filaments?
binds to the filament where ADP is high and destabilizes. Causes chunks to break off and treadmilling to increase in rate.
What are the four steps involved in actin mediated motility?
- Extension: leading edge moves out and forms lamellipodium
- New adhesions form
- Translocation - cell moves over
- Breaking of old adhesion
What does profilin do?
How is ARP (actin related protein) involved
mediating growth related to branched filaminets of actin.
ARP2/3 promotes branching. Branching generates the force needed to move the cell.
What does ARP have to bind in order to cause branching
NPF
Proteins polymerize into __________as the basis of cytoskeleton
dynamic filaments
Three levels of cytoskeleton
What is the most rigid type of cytoskeleton
microtubules
Two different forms of actin and growth patterns
Globular G-actin
Filamentous F-actin
growth occurs faster at + end than at - end.
What are the three steps of actin polymerization
- Nucleation *rate limiting
- Elongation
- Steady state
What is the role that ATP plays in actin polymerization
without ATP can’t’ depolymerize
How does the critical concentration compare at + and - end?
Cc is higher at - end so - end requires higher amount of G actin in order to polymerize.
What do actin binding proteins do?
Interact with the microfilaminets
Length: cofflin
Branching: Arp2/3
Cross-linking: Filamin
Motor Proteins: Myosin
Stability: CapZ and Tropomodulin
Organization: Nebulin
Steps in cell locomotion
- Extenstion to form lamellipodium
- New adhesions form
- Translocation
- Break old adhesions
Which part of a motor protein has the ATP binding (ATPase) activity?
Head domain
WHat ion is required for myosin movement along actin filaments?
Ca+2
Which step of ATP hydrolysis is the “power stroke” of vesicle transport?
Release of Pi
How do myosin II proteins combine to form thick filaments?
Aggregation of tail ends.
Muscle cell description
Muscle cells = muscle fibers
long multi-nucleated cells…1 cell = 1 fiber
Bundles of fibers contain actin and myosin II
Single sarcomere runs Z disc to Z disc
What causes muscle contractions?
Globular heads come off actin.
Move toward + end
Z discs contract
Happens in 100,000s of sarcomeres at once
What can cause multinucleated cells?
Dysfunction of myosin II
Role of intermediate filaments
Structural integrity to cells
Network in cells and @ tight junctions
Surround nucleus to form nuclear lamina (protect nucleus from sheer forces in cell).
Support organelles
Basic structure of microtubules?
hollow, rigid, polymers of tubulin
Functions of microtubules
Organization of organelles and transport vesicles
Beating of cilia and flagella
Structure of nerve cells, blood cells, and cilia / flagella
Chromosome alignment and separation
Compare / contrast kinesins and dyneins
Kinesins move to + end
Dyneins move to - end
Both move along microtubules
