Membranes, IC trafficking, protein sorting Flashcards
What are functions of cellular membranes?
- cell recognition + signaling
- compartmentation
- keep components of some metabolic pathways in place
- transport of materials
What are lipid rafts?
Function?
There is a special type of lipid rafts that are small invaginations. How do you call them?
areas in outer leaflet of lipid bilayer enriched in cholesterol, sphingomyelin + glycosphingolipids, stabilized through interactions w/ cytoskeleton
→ signal transduction
invaginations = caveolae
Which structures/organelles belong to the endomembrane system?
- nucleus
- endoplasmic reticulum (ER), endoplasmic reticulum - Golgi intermediate compartment (ERGIC), Golgi apparatus
- lysosome (phagosome, autophagosome), endosome, peroxisome, autophagic vacuole
- transport vesicles
- lipid droplets
What is a lysosome?
Function.
cell organelle responsible for breakdown of polymers
- contains acid hydrolases that break down almost everything
- V-ATPase generates a low pH of 5 inside the organelle (powered by ATP hydrolysis)
What are lipofuscin granules?
yellow-brown pigment granules composed of lipid-containing residues of lysosomal digestion
- one of the aging or “wear-and-tear” pigments
- especifically arranged around the nucleus
What happens with the lysosome?
fuses w/ primary endosome to form secondary endosome
What are the main functions of Golgi?
- processing of oligosaccharide chains
- sorting of proteins + delivery to intrac. destinations
- synthesis of sphingomyelin, glycolipids, proteogylcans, glucosaminoglycans
What are the functions of the ER?
- protein, lipid synthesis
- glucose production
- Ca2+ storage
What is the function of chaperones and chaperonins?
chaperones:
-
folding of proteins, ATPase activity
→ ADP, ATP binding until protein is folded - resp. for correct targeting of proteins to intracell. destinations
chaperonins:
barrel-like structures that enclose proteins until completed folding
Explain the cytosolic (non-secretory) pathway of protein sorting + related transport mechanism.
starts in cytosol when lacking ER signal sequence
either:
- mature cytosolic protein
- organelle specific
- mitochondria - transmembr. transp.
- peroxisome - transmembr. transp.
- nucleus - gated transp.
Explain the rER (secretory) pathway of protein sorting + related transport mechanism.
starts in cytosol if ER signal sequence
- guided to ER
- Golgi
-
cell surface (excreted)
- lysosome
- plasma membrane
-
cell surface (excreted)
all transported by vesicles
How do the 2 sorting pathways differ w/r/t to translation?
- cytoplasmic pathway = posttranslational
- ER pathway = cotranslational
Explain the mitochondrial protein sorting for matrix proteins.
- protein synthesized on cytosolic polyribosomes
→containpresequence (sim. to sign. sequ.) targeting matrix -
translocation through outer and inner mitochondrial membranes
- interaction with chaperones bc must be in unfolded state to pass through complexes
- presquence split off by maxtrix-processing protease (MPP)
- proteins refolded inside organelle
Explain the process of import of large molecules into the nucleus
since small molecules can easily diffuse through nuclear pore complexes (NPCs).
- cargo containing nuclear localization signal NLS binds to importin
- binds to NPC
- inactive Ran (GDP) translocates cargo + NLS through nuclear envelope
- Ran activated by GEFs → GTP, cargo released inside
- importins+ Ran recirculate to cytoplasm
How does nuclear export happen?
Differentiate btw 2 classes of molecules.
for not-mRNA molecules:
exportins guide cargo with nuclear export signals NES together w/ Ran through pores
for mRNA:
transported to cytoplasm as part of ribonucleoprotein mRNP w/o usage of Ran, but ATP needed
What is a preprotein?
protein w/ signal sequence
→ signal sequence removed
How are proteins directed to their destinations?
by signal sequences, either:
- signal peptide = sequence at end of protein
- signal patch = pocket formed by diff. polypeptide chains
→ bind to distinct signal recognition particle SRP consisting of RNA + 6 proteins
note function of individual proteins + Met amino terminal
Explain the process of ribosome coupling to the ER.
What is the fate of the proteins?
- SRP binds to signal sequence on nascent polypeptide chain on ribosome → elongation arrest
- SRP bound ribosome binds to SRP receptor protein on rER
-
SRP released, GTP → GDP + P
translation continues - translocation of protein through translocon into ER lumen
- signal peptidase detaches signal sequence + released back into cytosol, protein released into ER lumen
→ sorted
Which proteins are N-glycosylated in the ER?
Explain.
secretory proteins + soluble proteins destined for organelles distal to ER
- N-glycan chains added by oligosaccharide-protein transferase
- involves Asn side chains
How are ER membrane proteins incorporated?
steps 1-4 (cf. ribosome coupling to ER), then lateral transfer into membrane instead of release into ER lumen due to retention signals
What is a halt-/stop-transfer signal?
highly hydrophobic segment in transmembrane proteins → unfinished translocation → retention as transmembrane protein in the membrane
Differentiate btw transmembrane proteins.
Examples.
4 types
- type I: cross membrane once, N-terminal in ER lumen/cell exterior (ex: LDL receptor)
- type II: cross membrane once, C-terminal in ER lumen/cell exterior (ex: transferrin receptors)
- type III: sim. to type I, but no cleavable signal peptide (ex: cytochrome P450)
- type IV: cross membrane multiple times (ex: G proteins, glucose transporters)
How is the correct folding status of proteins in the ER monitored?
- by chaperones
- by calnexin: Ca2+ needed, in ER membrane
- by calreticulin: Ca2+ needed, not memb.-bound
- by protein disulfide isomerase (PDI): reshuffling of disulfide bonds
- by peptidyl prolyl isomerase (PPI): folding of prolin-cont. proteins
What happens with misfolded or incompletely folded proteins?
remain in ER, disposed by ER associated degradation (ERAD) if homeostasis perturbed = ER stress
What is the exact pathway of ERAD?
- ER stress (cf. ERAD) sensed + induces unfolded protein response (UPR)
→ transient inhibition of translation
→ incr. synthesis of degradation enzymes + ER chaperones - ubiquination of misfolded proteins
- retrotranslocation of proteins through ER membrane
- escorted to proteasomes by polyubiquitin-binding proteins
Explain the structure of mitochondria.
2 compartments: matrix + intermembraneous space (btw inner/outer membrane)
- outer membrane: porous, permeable
- inner membrane: impermeable, proteins for respiratory chain
What are the functions of mitochondria?
- oxidative phosphorylation in respiratory chain → produces most ATP for cell’s demands
- Ca2+ buffering + storage
- heat production
- catabolism of fatty acids
Describe the structure and function of the mitochondrial genome.
double stranded circular → heavy + light strand
⇒ 37 genes code for 13 proteins translated by mitochondrial ribosomes (no introns due to prokaryotic origin!)
Can mitochondrial diseases be inherited?
ONLY inherited maternally (oocyte provides all organelles, spermium only provides nucleus)
→ mostly associated w/ protein deficiency/production
Describe the different types of transport.
active: via ATP-driven pumps against gradient
passive: along gradient
- simple diffusion: higher to lower conc.
-
faciliated diffusion: higher to lower conc., but cannot pass through membrane normally due to size/hydrophilic character etc.
- via pumps
- via ion channels
Differentiate btw types of endocytosis.
Explain briefly.
- phagocytosis: macrophages/granulocytes ingest viruses, bacteria, cell debris
- pinocytosis: cellular uptake of fluid + fluid contents
- potocytosis: receptor-dependent in caveolae
- clathrin-mediated endocytosis: receptor-dependent
What are the requirements of endocytosis?
- E (usually hydrolysis of ATP)
- Ca2+
- microfilament system
Explain the process of exocytosis.
Fate of the “exocytozed” molecule?
- components snythesized in ER/Golgi put in vesicles
- hormone guides vesicle to cell-surface receptor
- local, transient change of [Ca2+]
-
fuses w/ plasma membrane →
- remains associated as membrane proteins
- become part of extracellular matrix
- signal other cells
Explain the formation of vesicles.
- initiation: coatomer proteins assemble
- invagination: membrane is budding
- constriction: coat proteins shape membrane into a sphere
- fission: vesicle is detached
- uncoating: coat dissasembles
Differentiate btw coatomers and for which vesicles they are apparent.
clathrin
- endocytosis
- regulated exocytosis: Golgi → plasma membrane
- Golgi → late endosome
COPI
- retrograde: Golgi → ER
- constitutive exocytosis: Golgi → plasma membrane
COPII
- anterograde: ER → Golgi
Explain the structure of clathrin.
3-limbed structure = triskelion
each limb: light + heavy chain
Explain the process of receptor-mediated endocytosis.
- assembly of clathrin to cargo receptors on plasma membrane directed by adaptins → coated pit
- bud formation
- dynamin (GTPase) binds → fission
- vesicle formation
- uncoating by hsc70 (ATPase)
What is the difference btw COPI/clathrin and COPII coated vesicles w/r/t their intracellular transport?
for COPII:
- Sar1 binds to plasma membrane
- GTP binds → activates Sar1 → budding initiated
⇒ later: Sar1 inactivated when GTP hydrolyzed → coat disassembly
for COPI/clathrin-coated vesicles:
- Arf instead of Sar1
What is the function of Rab GTPases?
vesicle targeting
- GEF: Rab·GDP in cytosol → Rab·GTP
- Rab·GTP binds to Rab effector proteins on vesicle→ tether to membrane
- fusion w/ membrane → GTP hydrolyzed, Rab·GDP released
What is the function of SNAREs?
initiate fusion
- v-SNARE on transport vesicle binds to complementary t-SNARE on target membrane
- 4-helix boundle
- ATPase (NSF) + αSNAP dissociate 4-helix boundle
What is the difference btw antero- and retrograde transport?
What is the importance of KDEL receptors?
- anterograde = ER → Golgi (COPII)
- retrograde = Golgi → ER (COPI)
BUT: KDEL receptors guide vesicles from ER to Golgi → there retained
What are G-proteins?
GTPases (activated when GTP bound) that transmit signals from outside stimuli to interior of a cell
What does colchicine do?
can be gained from autumn crocus
“mitotic poison” or spindle poison binds to tubulin and inhibits microtubule polymerization
What are the functions of the cytoskeleton?
Differentiate btw filaments.
- cellular protein scaffolding → structure and shape
- vesicular transport and cell division
3 filaments:
- microfilaments (actin)
- intermediate filaments
- microtubules
Differentiate btw types of actin.
Function?
- 3 types: α, β, γ
- β-actin: globular (G) subunit and filamentous (F) polymer (pointed and barbed ends polymerize)
function = actomyosin complex
- moving organelles, cellular motion during cell division
- cell adhesion structures
- maintenance of cell volume
Explain the reassembly of actin filaments
- at pointed end of actin filament: “old” actin·ADP bearing actin depolymerizing factor ADF cofilin disassembles to actin monomers
- actin·ADP → actin·ATP
- profilin directs actin monomers to barbed end
Which molecules are responsible for actin branching?
Arp2/3 complex
Which structures crosslink actin filaments?
Explain the structure of a microtubule.
Function?
polymer of tubulin subunits (α + β) → protofilmanets, 13 form→ microtubule
- polarized polymerization (- and + ends)
function:
- organelle movements
- mitotic spindle
- cilia and flagella
Explain the term dynamic instability w/r/t microtubules.
Why is this behavior important?
at + end
- A: intermediate cc of tubulin → frayed end in slow growing filaments
- B: high free GTP-tubulin dimer cc, hydrolysis outpaced → rigid end GTP-cap, rapid assembly
- C: GTP hydrolysis weakens the tubulin dimer interaction → protofilaments rapidly disassemble
⇒ growth from centrioles, ability to direct intrac. movement
Differentiate btw dyneins.
In which direction do dynein and kinesin walk on the microtubule?
cytosolic dynein → + end (axonemal dynein in flagella)
kinesin → - end
What are the properties of intermediate filaments?
Classes?
- no polarity
- no motor proteins associated
- high tensile strength, resistant to compression, twisting and bending forces
- heterogeneous
