test #1 Flashcards
explain: hydrophobic AA (location, properties, categories)
- inside core of soluble prot.
- usually have non-polar side chains
- water insoluble
- unable to form H-bonds
- 2 categories
1. aromatic
2. aliphatic
name: hydrophobic AA (by category) (3) (5)
- aromatic
⤷ phenylalanine
⤷ tyrosine
⤷ tryptophan - aliphatic
⤷ alanine
⤷ valine
leucine
⤷ isoleucine
⤷ methionine
**tyrosine has hydroxyl so it’s both phobic and phatic
explain: hydrophilic AA (location, properties, categories)
- on surface of prot.
- can be charged or uncharged
name: hydrophilic AA (by category) (2) (2) (2) (2)
- +ive charged
⤷ lysine
⤷ arginine - -ive charged
⤷ aspartic A, glutamic A - uncharged w/ polar hydroxyls
⤷ serine
⤷ threonine - uncharged w/ polar amine groups
⤷ asparagine
⤷ glutamine
name + explain: special AA (4)
- cysteine
- forms covalent bonds w other cysteines (the S atom)
- bonds = disulfide bridge - proline
- R group forms covalent bond w/ amino group of AA
- causes kink in polypep. - glycine
- very small
- can tuck into folding prot. making bend - histidine
- amino diethyl side chain (imadazole)
name: protein organization lvls + shapes
- primary = linear
- secondary = α helix, β sheets, turns and loops
- tertiary = 3d arrang. of single polypep.
- quaternary = conformation of multiple prot. into prot. complex
explain: primary prot. struc.
- polypep. naturally folding = random coil
- stabilized struc. = statistical coil
explain: secondary prot. struc.
- α helix = spiral
⤷ H-bonds w/ 4 AA away to make helical struc.
⤷ R-groups can change phobic/philic properties (amphi = opp. sides) - β sheets = planar alignment of 2+ strands
⤷ H-bonds w/ adjacent strands
⤷ can be parallel or antiparallel
⤷ R-groups can change phobic/philic properties (amphi = opp. faces of sheet) - turns/loops = connectors
⤷ bends, turns
⤷ H-bond between non-variable AA side chain, amino group, carboxyl group
⤷ 2 special AAs
⤷ proline at 2 -> sharp bend in polypep.
⤷ glycine R group at 3 -> minimizes steric hindrance
explain + name: motifs
- for secondary prot. struc.
- unique collection of struc.
- coiled-coil
- zinc-finger
- barrel
- helix-loop-helix
explain: coiled-coil motif
- 2 α helices wrapped around each other
- possible bc both helices amphipathic
- phobic surfaces aren’t all on the same side
⤷ phobic surface spirals around each helix - ex. leucine zipper
- in DNA bc shape fits well
explain: zinc finger motif
- α helix + 2 β strands
- held by Cys or His in specific spots
- conserved shape + struc.
- in DNA binding prot.
explain: β barrel motif
- large β sheet looping back on itself
- 4 - 10 anti-parallel strands
- first and last strands H-bond
- in channels/pores of phobic membranes
- barrel = amphipathic
⤷ outside = phobic, inside = philic
explain: helix-loop-helix
- 2 small α helices
- held by non-covalent interactions between specific AA
- shape dep. on calcium cofactor
explain: tertiary prot. struc. (define domain)
- domain: substruc. prod. by any part of polypep. that can fold indep. into compact stable struc.
⤷ func. domains - reg. of prot. that perform certain activities
⤷ struc. domains = reg. of prot. assoc. w/ recog. shape/charac. - ex. Src prot.
⤷ has 4 diff. domains
explain: quaternary prot. struc.
- ex. dimer/trimer, homodimer, heterodimer
- homodimer: 2 identical polypep. w/ same primary seq. and same tertiary struc.
- heterodimer: 2 prot. diff. from each other
- ex. haemoglutin
⤷ prot. of influenza virus
⤷ tertiary = 2 domains (globular and fibrous)
⤷ quaternary = trimer
define: intrinsically unstructed prot.
- lack tertiary struc. as isolated subunits
- ex. zinc fingers
⤷ unstruc. w/out substrate
⤷ DNA substrate interact w/ finger -> stabilized -> func. zinc finger
name: post-translational modifications (7)
- acetylation
- methylation
- phosphorylation
- hydroxylation
- carboxylation
- glycolysation
- lipidation
explain: acetylation
- adding acetyl
- protects from protease degradation
- can change activity of prot.
- reversible
explain: methylation
- adding methyl
- changes gene expression
- reversible
- ex. histidine -> 3-methyl histidine
explain: phosphorylation
- transfer of phosphate from ATP -> -OH of serine, tyrosine, or threonine by kinases
- kinase catalyzes rxn
- dephosphorylation by phophatase
- activate/deactivate prot. by changing shape or ability of prot. to interact w/ substrate
explain: hydroxylation
- adding hydroxyl (OH)
- important for changing struc. of prot.
- ex. triple helical coiled-coil of collagen
⤷ need hydroxylation to associated 3 helices
explain: carboxylation
- adds -ive charge -> changes properties
- ex. glutamate -> gammacarboglutamate
⤷ 1 -ive charge -> 2 -ive charges
⤷ facilitates ionic bond formation and allows +ive cofactor to bind
explain: glycolysation
- adding carbs (sugars to specific R groups)
- for proper protein folding + protects prot. from proteolysis
explain: lipidation
- adds lipids
- anchors prot. to hydrophobic biomembranes
define: native state
- most func. conformation of prot.
- most thermodynamically stable
name: rules of folding (3)
- spontaneous
- reversible
- unique
explain: reversible denaturation experiment (anfinsen)
- ribonuclease A prot.
- cysteine bonds and non-covalent interactions hold prot. in tertiary
- denature by:
⤷ using urea to break H-bonds and disrupt phobic interactions
⤷ using beta mercaptoethanol to break disulfide bridge - denature prot. have same primary struc.
- reassemble by:
⤷ dialysis to remove denaturants
⤷ prot. refolds itself - proves prot. folding is spont., reversible, unique (bc returned to original prot.)
explain: villin prot. folding
- prot. folding = random trial and error
- keeps un/refolding to reach most thermodynamically stable conformation
- villin C term. = spont. folding 3 helix bundle
explain: example to show importance of prot. folding
ex. sickle cell anemia
- hemoglobin = 4 subunits
⤷ 2 native config. - affected = single AA change from glutamate to valine in position 6
- changes shape
⤷ from tetramers to long polymers - still carries O2
- misshapen RBC get stuck in capillaries -> blockages -> not able to carry O2 to all organs -> anemia
define: chaperone
- prot. that prevent inappropriate interaction between AA
- increases efficiency of prot. folding
- 2 types
1. molecular chaperones
2. chaperonins
explain: molecular chaperones w/ example
- monomeric prot.
- prevents incorrect folds due to phobic interactions
- ex. heat shock protein (Hsp)
⤷ expressed at high lvls of stress
⤷ Hsp70: in cyto. and mito. of euk. cells
⤷ BiP: in ER
⤷ DnaK: in bac.
explain: Hsp70
- 2 domains
1. nucleotide binding domain
2. substrate binding domain - nuc. binds ATP for E
- phobic AA on Hsp70 allows it to bind to patches on unfolded prot.
- ATP hydrolyzes to ADP (stimulated by co-chap.)
⤷ changes conformation of HSP70 chap.
⤷ changes shape of prot. allowing correct folding - ADP released from Hsp70
⤷ assisted by nuc. exchange factor (GrpE or BAG1) - folded prot. released
- new ATP fills domain
explain: chaperonins
- large macromolecule complexes
- collection of prot. in complex form chamber/barrel
⤷ barrel = place for unfolded prot. to move into to fold in isolation - ex. TCiP: in euk. cyto
- ex. GroEL: in bac. and chloroplasts
- ex. Hsp60: in mito.
explain: chaperonin complex (of bacteria)
- 2 large GroEL subunits
- tops of chambers alternate open/closed
⤷ capped by GroES small subunit
explain: chaperonin func.
**only 1 GroEL helping at any given time
- bottom chamber releases GroES cap and ADP while top chamber binds to ATP and new substrate peptide
- new GroES cap binds to top of GroEL
⤷ closes chamber and isolates GroEL - confromational change enlarges chamber dimensions
- ATP hydrolysis allows GroES to come off
⤷ allows prot. to diff. out
explain: GroEL chamber struc.
- 3 domains
- each Hsp60 binds to 1 ATP mol.
- 7 Hsp60 form wall of GroEL
explain: protein degradation
- sometimes proteins don’t fold
⤷ can aggregate - can also degrade:
⤷ denatured prot.
⤷ prot. at too high conc.
⤷ prot. endocytosed into cell
⤷ regulated prot. (cyclically made and degraded)
explain: steps of protein degradation
- prot. = tagged by small prot. ubiquitin to specific AA in polypep. seq.
- tag = recog. by proteolytic machinery (proteasome)
⤷ prot. cleaved into short pep. seq.
⤷ eliminated prot. func.
explain: ubiquitinylation
- attachment of ubiquitin to target prot.
- req. 3 enz.
1. E1: ubi. activating enz.
⤷ recognizes free ubi. prot. in cyto + picks it up
- E2: ubi. conjugating enz.
⤷ facilitates attachment to prot. - E3: ubi. ligase
⤷ recog. specific target + attaches ubi.
explain: proteasome struc.
- like chaperonin
- walls = subunits to form barrel w/ caps
- barrel contains proteolytic enz.
⤷ breaks down any prot. in barrel
explain: proteasome func. w/ example
- ubi. tag recog. by proteasome cap
- prot. enters cap
- unfolds (cleaved)
⤷ ubi. tag removed first - further degraded by cytosolic proteases or in lysosome
- ex. spinocellular atazia
⤷ unfolded Ataxin prot. cannot be unfolded
⤷ gets stuck on proteasome -> aggregate
question + define: what do prot.-ligand interactions depend on? (2)
- specificity
⤷ ability to preferentially bind to 1/small # of mol. and not others - affinity
⤷ strength of binding
⤷ strong = bond for long time
⤷ weak = fall apart immediately
**dep. on molecular complementarity
define: molecular complementarity (stable + less stable)
- shapes of mol. fit together well enough -> favourable non-covalent interaction forms
- stable
⤷ lots of H-bonds, ionic bonds, van der whaals
⤷ bc shapes are close - less stable
⤷ 2 -ives facing each other
⤷ no ionic bond
⤷ shapes not complementary so they can’t be close to form interaction
explain: ligand-binding pocket (cAMP)
- ligand binding site = pocket
- for prot. interactions w/ mol. other than prot.
- cAMP = regulatory molecule
⤷ can modify prot. func. - 6 AA help cAMP bind to pocket
explain: binding affinity
- free E of interaction between prot. + ligand
- binding affinity = K (association constant) or Keq. (equilibrium)
- high K = tend to R = high affinity
- low K (high Kd) = tend to L = low affinity
**Kd = dissociation constant
explain: enzymes
- prot. that cat. mol. rxn
- lowers free E state = favourable bc stable
- enz. decrease free E of transition state
- enz. binds ligands to promote chem. rxn
explain: enz. active sites
- 2 regions
1. binding site/pocket = determines specificity
2. catalytic site = promotes rxn - needs high specificity and high affinity
define: Vmax and Km
- Vmax = max. velocity of rxn
- Km (michaelis constant) = affinity between subs. and enz.
**fixed amount of enz. will reach same Vmax
⤷ no matter affinity
just needs more subs. if low affinity
explain: prot. kinase A (PKA) (in regulating/modifying prot. func.)
- enz. prot. (kinase enz.)
- adds phosphate to target prot.
- 2 substrates bind
1. target prot.
2. nucleotide ATP - 2 domains form nuc. subs. binding pocket
⤷ 1 for ATP, 1 for target prot. - PKA changes shape when bound
- binding site = specific for ATP
- target prot. regoc. by glutamic A in large domain
explain: confomational changes in PKA
- open conformation -> target pep. binds to PKA
⤷ binding sites = exposed - closed conformation -> large + small domains move together
⤷ glycine lid traps substrate
⤷ allows phosphate to transfer ATP to target protein - phosphorylated pep. and ADP have lower affinity to PKA sites
⤷ so sites open and they leave
name: mechanisms for regulating protein func. (4)
- allosteric regulation
- covalent modification
- proteolytic cleavage
- protein complexes
explain: allosteric regulation
- for regulating prot. func.
- mods. by binding of effector mol. at site other than active site
- usually conformational change in protein shape
- effector molecule = allosteric modulators
⤷ + = increases activity
⤷ - = decreases activity (inhibits)
explain: PKA and allosteric modification (active and inactive)
- PKA = allosteric enz.
- inactive PKA has 2 regulatory (R) subunits and 2 catalytic (C) subunits
⤷ quaternary/tetrametric struc. (4 subunits) - inactive bc binding site blocked by domain on R
- cAMP = +ive allosteric activator for PKA
⤷ binds to R leading to conformational change - changing subunit -> changes pseudosubstrate -> no bind to C -> active PKA
**low conc. cAMP -> PKA inactive (vv)
explain: allosteric inactivation (allosteric regulation)
- ATCase = aspartate transcaramoylase
⤷ regulated by allo. inhibitor CTP - CTP binds to regulatory subunits -> conformation change -> complex twists into inactive.tense conformation
- vv = low CTP -> binding sites empty -> relaxed state
explain: negative feedback loop of allosteric inactivation (allosteric regulation)
- turning off pathway when you don’t need the product by using the product as a signal
- ex. CTP
⤷ -ive modulator
⤷ if CTP conc. = high -> cell doesn’t need more
⤷ presence of CTP = prevents CTP overproduction
explain: cooperative allostery (allosteric regulation)
- binding of ligand to one subunit
- causes change in affinity of all subunits
explain: example of cooperative allostery (allosteric regulation)
ex. hemoglobin
- high O2 affinity in lungs, low O2 affinity in tissues
- 2 α subunits, 2 β subunits
- 2 states (T/inactive = low O2 affinity, R/active = high O2 affinity)
- affinity changes when 1 O2 molecule binds
- increases efficiency
explain: covalent modification (examples)
- for regulating prot. func.
- sometimes reversible
- ex. phosphorylation, acetylation, methylation, carboxylation
explain: phosphorylation (covalent modification) w/ example
- kinase prot. CDK phophorylates other prot.
- inactive CDK can’t bind to subs. bc blocked
- active CDK: block moves after being phosphorylated
- increases -ive charge bc phosphate
- ex. R-OH
⤷ before: kinase uses ATP to covalently attach phosphate (inactive)
⤷ after: phosphatase removes phosphate (active)
explain: proteolytic cleavage (for regulating prot. func.)
- irreversible
⤷ bc can’t reform broken pep. bonds - cell makes a lot of prot. -> cleaves at specific points to activate it
- changes create substrate binding domain
explain: protein complexes (for regulating prot. func.)
- associating enz. to work together
- substrate binds to A -> prod. intermidiates
- intermediates bind to B -> prod. more intermediates
- intermediates bind to C -> prod. product
- slows overall rate of pathway
- 2 methods to limit the effects of the slow
1. multimeric complex (enz. bind together)
2. adding a scaffold for enz. to bind to
list: steps to isolate a protein (7)
- assay (unique)
- select prot. source
- extract prot. from source
- solubize prot.
- stabilize prot.
- fractionate prot.
- determine purity (using assay)
explain: solubility (in terms of isolating a prot.) (soluble vs insoluble, factors that affect it)
- soluble
⤷ cytosolic
⤷ secreted - insoluble
⤷ transmembrane (bc amphipathic)
⤷ mem. assoc. prot. - affected by:
⤷ solution pH
⤷ salt conc.
⤷ presence of detergents (can stabilize molecular interac. in insoluble prot. making them more soluble)
explain: stabilization (in terms of isolating a prot.) (how, factors that affect it)
- trying to maintain native struc. to prevent degradation
⤷ pH, salt conc., presence of co-factors help - increase temp. -> denature
- maintaining non-covalent interactions helps stabilize
- factors:
⤷ pH
⤷ temp.
⤷ protease inhibitors
⤷ ligands
⤷ salts
⤷ concentration
name: fractionation techniques (4)
- charge
- size
- polarity
- specificity of binding
explain: differential centrifugation
- spin tubes at 1000g
⤷ pellet contains nuc. and chloro. - spin at 10000g
⤷ pellet contains mito. - spin at 100000g
⤷ microsomal fraction (ER, golgi, lyso., peroxi.) - spin one more time
⤷ supernatant (cyto., soluble prot.)
explain: relationship between wavelength, distance, and resolution in microscopy
- lower wavelength = lower distance = better resolution
- shorter wavelength = better resolution
explain + describe: brightfield microscopy
- live/fixed sample
- stained or unstained
- can see indiv. cells
my description
- the colourful one that’s not red or green
explain + describe: phase contrast
- unfixed
- unstained
- transparent specimen
- high contrast image
- can see internal structure
- rely on enhancing difference in density
my description
- the blue-ish gray metally looking one
- its giving kpc blue metal vibes
explain + describe: nomarski/DIC
- live specimen
- differential interference contrast
- clearer, sharper image of edges
- rely on enhancing difference in density
my description
- the boring gray one
- look like 3d bumpy guys
explain + describe: immunofluorescence microscopy
- location of specific molecule in cell
- mol. tagged w/ dyes or fluorescent antibodies
- primary antibody = locates target prot.
- secondary antibody (covalently attached to fluorophores) = recog. primary antibody
my description
- glowy green and blue
- not stringy
explain + define: confocal scanning microscopy
- higher resolution images
- only excites fluorophores (so it’s clearer)
my description
- stringy clear red, green, blue glowy
explain + define: TEM and SEM
TEM = transmission electron microscopy
- beam directed onto specimen
my description
- very detailed gray
SEM = scanning electron microscope
- beam on metal -> 3d image
- more detail
my description
- yellow salt fossil looking thing
name: purpose of biomembranes (4)
- defines boundaries
- selectively permeable
- holds prot. that can mediate cell-cell interac.
- flexible and dynamic w. shape of cell
explain: FRAP
- fluorescence recovery after photobleaching
- fluorescent = tag
- too much exposure -> bleached
- shows fluidity bc bleached prot. get dispersed around mem. again making it fluorescent (but less bright)
name + explain: factors affecting mem. fluidity (and how they affect it ex. increase or decrease) (3)
- lipid composition
- saturated (straight) = decrease fluidity
⤷ pack together more tightly
- long chains = decrease fluidity
⤷ pack together more tightly - cholesterol
- presence = decrease fluidity
⤷ packs the chains more
- absence = too fluid + permeable
- ex. animals that hibernate
⤷ high conc. of cholesterol -> separates phospholipids so chains can’t crystallize - temperaturee
- low = decrease fluidity
- high = increase fluidity
- ex. bacteria
⤷ cleave chains at low T to help fluidity
- ex. cold blooded animals
⤷ add more cholesterol in resp. to cold T
- ex. hibernating animals
⤷ increase unsaturated (bent) chains in preparation for decrease in T
explain: lipid rafts
- region of high cholesterol
- taller than rest of mem.
- makes mem. less fluid
- has 2 leaflets
- part of the mem. not just on top
explain: single pass TM and multipass TM (integral proteins)
- single pass = crosses mem. once
- multipass = passes mem. many times
explain: beta-barrel (integral protein)
- exterior = phobic
- interior = philic
- forms philic pore through phobic membrane
explain: channels (integral protein)
- collection of α helices
- exterior = phobic
- interior = philic
explain: lipid anchored prot.
- assoc. w/ one leaflet by covalently attached lipid modifications
- mod. prot. by ex. acetylation (add lipid anchor to N) + prenylation (add lipid anchor to C)
explain: peripheral prot.
- interac. w/ other prot. that are embedded/anchored to mem.
- indirectly attached to mem.
- can often reversibly attach and detach from mem.
define: peroxisome (role)
- small oval w. single bilipid mem.
- no genetic info
- rep. by fission
explain: peroxisome biogenesis
- peroxisome prot. synth. happens in cyto. then moved to peroxi.
- peroxi. mem. prot. are targeted to precursor
⤷ makes peroxisomal ghost - once prot. are in mem.: used to transport peroxi. matrix prot. into organelle
explain: luciferase
- enz. that allows bioluminescence
- in fireflies
name: rules to prot. transport (5)
- signal sequence on transported prot.
- receptor for signal sequence
- translocation channels
- require E
- way to target prot. to specific and different locations in organelle
explain: rule 1 of prot. transport in peroxisomes
PEPTIDE SIGNAL SEQUENCE
- sig. seq. on luciferase is also on peroxisomes
⤷ PTS1 (peroxisomal-transport seq. 1)
- PTS1 at C term. of translated peroxi. prot.
- after translation: peroxi. prot. fold and leave C term. visible
question: is PTS1 necessary and sufficient in peroxisomes?
- necessary hypothesis: no PTS1 means luciferase will no longer go into peroxi.
⤷ hypo = true (bioluminescence all over cell not just in peroxi. when no PTS1)
YES NECCESSARY - sufficient hypothesis: if PTS1 sufficient, DHFR will enter peroxi.
⤷ DHFR = cytosolic prot.
⤷ w/ PTS1: DHFR in peroxi. (matches catalase spots)
YES SUFFICIENT
explain: rule 2 of prot. transport in peroxisomes
SIGNAL RECEPTOR
- Pex5 = cytosolic prot.
⤷ recog. PTS1 seq.
- Pex5 binds to PTS1 at C term
⤷ associates w/ Pex14 to bring prot. to peroxi. mem.
explain: rule 3 of prot. transport in peroxisomes
TRANSLOCATION CHANNEL
- Pex14 forms translocon
- once Pex14 gets Pex5 and prot. into peroxi, Pex5 dissociates
explain: rule 4 of prot. transport in peroxisomes
E REQUIREMENT
- ubiquitinoylation requires ATP hydrolysis
- Pex5 gets ubi. and deubi. to release from matrix of peroxi. and get recycled
explain: rule 5 of prot. transport in peroxisomes
PROT. SORTED INTO DIFF. COMPARTMENTS
- 2 possible places for peroxi.
1. mem.
2. matrix
question: what is the consequence of a defect in transport to the peroxisome?
- zellweger’s syndrome
- cells accumulate into very long chain
- disrupts neuronal movement and brain dev.
question: do mitochondria have post-translational transport?
- hypo: if fully translated prot. is transported, prot. in presence of mito. will move into mito.
- exp.: mito. prot. synth. in cell-free sys.
- add mito. and then protease
⤷ prot. safe bc moved into mito. - add only protease
⤷ prot. degraded
explain: rule 1 of prot. transport for mitochondria
SIGNAL SEQUENCE
- has a matrix targeting motif at N term
- amphipathic alpha helix
question: is helix necessary and sufficient in mito.?
- necessary hypothesis: if helix = disrupted, mito. prot. won’t be targeted to mito.
⤷ hypo = true
YES NECCESSARY - sufficient hypothesis: if helix added to GFP, it will be targeted to mito.
⤷ hypo. = true bc GFP no longer in cyto.
YES SUFFICIENT
explain: rule 2 of prot. transport for mito.
SIGNAL RECEPTORS
- recog. by import receptors in outer mem. of mito.
- amphi. helix of motif fits into phobic binding pocket of receptor
explain: rule 3 of prot. transport for mito.
TRANSLOCATION CHANNEL
- general import pore in outer mem. aka translocon Tom40
- matrix-targeting seq. binds to import recept. -> translocated to general import pore
- if targeting to matrix:
⤷ continues to another translocon of inner mem.
- at contact sites: the 2 translocons align for direct mvt. of prot. w/ matrix targeting motif through both openings
explain: rule 4 of prot. transport for mito.
E REQUIREMENT
- mito. prot. = translated in cyto.
⤷ grabbed by Hsc70 (chap.)
- grabs port. to prevent going backwards in translocon
- Hsc70 needs ATP
- ATP also drive conformational change in Hsc70
⤷ pully polypep. into matrix
explain: rule 5 of prot. transport for mito
PROT. SORTED INTO DIFF. COMPARTMENTS
- possible places = mito. matrix and mem.
- targeting to mem. needs:
⤷ N term matrix targeting motif
⤷ stop-transfer seq.
- targeting to matrix = same steps as for mem.
⤷ N term motif recog. by import receptor
⤷ N term translocated into matrix
- stop-transfer seq. forms phobic alpha helix
⤷ 2 tasks: stop translocation and directs transfer of polypep. out of translocon into inner mem.
question: is stop-transfer sequence necessary and sufficient in mito. prot. transport?
- removing STS -> prot. still entering matrix due to matrix-targeting seq. but can’t get into mem.
YES NECESSARY FOR MEM, NO NECESSARY FOR MATRIX - NO SUFFICIENT FOR MEM
- need both STS and matrix targeting seq.
question: is unfolded prot. necessary and sufficient in mito. prot. transport?
ex. DHFR
- necessary hypo: folded = won’t be transported
- tagged w. matrix-targeting motif
- in presence of Hsc70, DHFR stay unfolded (can be transported into mito. matrix)
YES SUFFICIENT
- adding methotreate = maintains folded DHFR
- matrix-targeting seq. still successful into entering mito.
- folded can’t enter translocons
YES NECESSARY
question: are prot. targeting while translation is active (RER)?
- hypo.: if transport during translation, prot. will exit ribo. and go directly into microsome
- microsomes mimic ER bc same mem. assoc. prot.
- add detergent + protease -> prot. digested
- no detergent + protease -> newly synth. prot. protected inside micro.
question: do translocation and translation need to happen at the same time (RER)?
- hypo.: if translation happens, import to ER can’t happen
- in vitro w/out micro. -> prot. cant enter micro
⤷ bc fully translated - in vitro w/ micro. -> mature prot. in micro.
- import must occur co-translationally
explain: rule 2 of prot. transport of RER
SIGNAL RECEPTOR
- signal recognizing particle (SRP)
- binds to ER sig. seq. (on N) and large ribo. subunit
⤷ stalls translation
- SRP needs SRP receptor on mem.
- SRP recep. = transmem. dimer
explain: rule 1 of prot. transport of RER
PEPTIDE SIGNAL SEQUENCE
- domain at N term (short)
- used to target prot. to ER
- prot. synth. happens N to C so sig. seq. gets prod. first
explain: rule 3 of prot. transport of RER
TRANSLOCATION CHANNEL
- translocon opens as nascent prot. transferred into interior of translocon
- SRP dissociated form sig. seq. during translocation
- ribo. stay assoc.
- as AA added in translation, prot. is pushed through translocon
explain: rule 4 of prot. transport of RER
E REQUIREMENT
- SRP and SRP recep. have GTP activity
- powers transfer of pep. into translocon
explain: struc. of ribosome translocon complex
- large ribo. subunit interacts w/ translocon
⤷ leaves little/no space that exposes nascent prot. - ribo. associates w/ translocon
⤷ makes RER rough - ribo. docks at translocon
- N term enters lumen -> sig. seq. cleaved by TM prot. signal peptidase
- translation complet -> polypep. entirely pushed into lumen
explain: rule 5 of prot. transport of RER
PROT SORTED INTO DIFF. COMPARTMENTS
- prot. w/ ER sig. seq. = soluble = targeted to ER lumen
- TM prot. = targeted to ER mem.
- use second sig. seq. to embed prot. into mem.
- topogenic seq.: needed to embed prot. into ER mem. during co-translational transport
⤷ determines topology of prot. (# of times it crosses mem.)
explain: type I integal prot. (ER)
- 2 signals
- uses same N term as ER prot.
- stop-transfer anchor (STA) forms phobic alpha helix
- stops translocation through translocon -> transfers prot. to mem. -> anchors prot.
- SRP recog. N term and threads through translocon
⤷ prot. pushed out happens same time as translation - STA translated and folds into helix
⤷ stops translocation + sends sig. to make translocon open - allows topo. seq. to diffuse into surrounding mem.
explain: type II and III integral prot. (ER)
- II: N on cyto, C on luminal
- III: N on luminal, C on cyto.
- both lack N term sig seq.
- both have signal-anchor seq.
II
- SRP recog. SA seq. to bring nascent prot. and assoc. ribo. to ER mem
- SA sig. transferred to translocon
- translocon opens laterally letting SA seq. diffuse out
- +AAs keep N term from transferring to translocon
- C term pushed through translocon into lumen
- final prot. anchored by single phobic TM domain
III
- SRP recog. SA seq. to bring nascent prot. and assoc. ribo. to ER mem
- SA sig. transferred to translocon
- translocon opens laterally letting SA seq. diffuse out
- +AAs keep seq. near SA seq. in cyto
- new prot. pushed out
- final prot. anchored by single phobic TM domain
**has short N term so threading into translocon = easier
explain: type IV integral prot. (ER)
- passes multiple times
⤷ each pass = topo. seq. - 2 types
⤷ IV A = N term on luminal side (SA II: N on cyto SA III: N on luminal)
⤷ IV B = N on luminal
explain: hydropathy profiles
- graphicl rep. of all AA on polypep.
- peak immediately at N tem = ER sign seq.
- middle peak = STA seq.
- II = longer III = shorter (for ER)
- many peaks suggests topo. seq. (multipass)
