Membranes, IC trafficking, protein sorting

Question 1

What are functions of cellular membranes?

Answer 1

• cell recognition + signaling
• compartmentation
• keep components of some metabolic pathways in place
• transport of materials

Question 2

What are lipid rafts?

Function?

There is a special type of lipid rafts that are small invaginations. How do you call them?

Answer 2

areas in outer leaflet of lipid bilayer enriched in cholesterol, sphingomyelin + glycosphingolipids, stabilized through interactions w/ cytoskeleton

→ signal transduction

invaginations = caveolae

Question 3

Which structures/organelles belong to the endomembrane system?

Answer 3

• nucleus
• endoplasmic reticulum (ER), endoplasmic reticulum - Golgi intermediate compartment (ERGIC), Golgi apparatus
• lysosome (phagosome, autophagosome), endosome, peroxisome, autophagic vacuole
• transport vesicles
• lipid droplets

​

Question 4

What is a lysosome?

Function.

Answer 4

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)

Question 5

What are lipofuscin granules?

Answer 5

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

Question 6

What happens with the lysosome?

Answer 6

fuses w/ primary endosome to form secondary endosome

Question 7

What are the main functions of Golgi?

Answer 7

• processing of oligosaccharide chains
• sorting of proteins + delivery to intrac. destinations
• synthesis of sphingomyelin, glycolipids, proteogylcans, glucosaminoglycans

Question 8

What are the functions of the ER?

Answer 8

• protein, lipid synthesis
• glucose production
• Ca²⁺ storage

Question 9

What is the function of chaperones and chaperonins?

Answer 9

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

Question 10

Explain the cytosolic (non-secretory) pathway of protein sorting + related transport mechanism.

Answer 10

starts in cytosol when lacking ER signal sequence

either:

• mature cytosolic protein
• organelle specific
  
  • mitochondria - transmembr. transp.
  • peroxisome - transmembr. transp.
  • nucleus - gated transp.

Question 11

Explain the rER (secretory) pathway of protein sorting + related transport mechanism.

Answer 11

starts in cytosol if ER signal sequence

1. guided to ER
2. Golgi
3. • cell surface (excreted)
     - lysosome
     - plasma membrane

​all transported by vesicles

Question 12

How do the 2 sorting pathways differ w/r/t to translation?

Answer 12

• cytoplasmic pathway = posttranslational
• ER pathway = cotranslational

Question 13

Explain the mitochondrial protein sorting for matrix proteins.

Answer 13

1. protein synthesized on cytosolic polyribosomes
   →containpresequence (sim. to sign. sequ.) targeting matrix
2. translocation through outer and inner mitochondrial membranes
   
   • interaction with chaperones bc must be in unfolded state to pass through complexes
3. presquence split off by maxtrix-processing protease (MPP)
4. proteins refolded inside organelle

Question 14

Explain the process of import of large molecules into the nucleus
since small molecules can easily diffuse through nuclear pore complexes (NPCs).

Answer 14

1. cargo containing nuclear localization signal NLS binds to importin
2. binds to NPC
3. inactive Ran (GDP) translocates cargo + NLS through nuclear envelope
4. Ran activated by GEFs → GTP, cargo released inside
5. importins+ Ran recirculate to cytoplasm

Question 15

How does nuclear export happen?

Differentiate btw 2 classes of molecules.

Answer 15

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

Question 16

What is a preprotein?

Answer 16

protein w/ signal sequence

→ signal sequence removed

Question 17

How are proteins directed to their destinations?

Answer 17

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}

Question 18

Explain the process of ribosome coupling to the ER.

What is the fate of the proteins?

Answer 18

1. SRP binds to signal sequence on nascent polypeptide chain on ribosome → elongation arrest
2. SRP bound ribosome binds to SRP receptor protein on rER
3. SRP released, GTP → GDP + P
   translation continues
4. translocation of protein through translocon into ER lumen
5. signal peptidase detaches signal sequence + released back into cytosol, protein released into ER lumen

→ sorted

Question 19

Which proteins are N-glycosylated in the ER?

Explain.

Answer 19

secretory proteins + soluble proteins destined for organelles distal to ER

• N-glycan chains added by oligosaccharide-protein transferase
• involves Asn side chains

Question 20

How are ER membrane proteins incorporated?

Answer 20

steps 1-4 (cf. ribosome coupling to ER), then lateral transfer into membrane instead of release into ER lumen due to retention signals

Question 21

What is a halt-/stop-transfer signal?

Answer 21

highly hydrophobic segment in transmembrane proteins → unfinished translocation → retention as transmembrane protein in the membrane

Question 22

Differentiate btw transmembrane proteins.

Examples.

Answer 22

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)

Question 23

How is the correct folding status of proteins in the ER monitored?

Answer 23

• 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

Question 24

What happens with misfolded or incompletely folded proteins?

Answer 24

remain in ER, disposed by ER associated degradation (ERAD) if homeostasis perturbed = ER stress

Question 25

What is the exact pathway of ERAD?

Answer 25

1. ER stress *(cf. ERAD)* sensed + induces **unfolded protein response (UPR)** → transient inhibition of translation → incr. synthesis of degradation enzymes + ER chaperones 2. **ubiquination** of misfolded proteins 3. **retrotranslocation** of proteins through ER membrane 4. escorted to **proteasomes** by **polyubiquitin-binding proteins**

Question 26

Explain the structure of mitochondria.

Answer 26

2 compartments: **matrix + intermembraneous space** (btw inner/outer membrane) * **outer membrane:** porous, permeable * **inner membrane:** impermeable, proteins for respiratory chain

Question 27

What are the functions of mitochondria?

Answer 27

* **oxidative phosphorylation** in respiratory chain → produces most ATP for cell's demands * **Ca²⁺ buffering + storage** * **heat production** * **catabolism of fatty acids**

Question 28

Describe the structure and function of the mitochondrial genome.

Answer 28

double stranded circular → **heavy + light strand** **⇒ 37 genes** code for **13 proteins** translated by mitochondrial ribosomes (no introns due to prokaryotic origin!)

Question 29

Can mitochondrial diseases be inherited?

Answer 29

**_ONLY_ inherited maternally** (oocyte provides all organelles, spermium only provides nucleus) → mostly associated w/ protein deficiency/production

Question 30

Describe the different types of transport.

Answer 30

**_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**

Question 31

Differentiate btw types of endocytosis. Explain briefly.

Answer 31

* **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

Question 32

What are the requirements of endocytosis?

Answer 32

* **E** (usually hydrolysis of ATP) * **Ca²⁺** * **microfilament system**

Question 33

Explain the process of exocytosis. Fate of the "exocytozed" molecule?

Answer 33

1. components snythesized in ER/Golgi put in **vesicles** 2. **hormone** guides vesicle to cell-surface receptor 3. local, transient change of **[Ca²⁺]** 4. **fuses w/ plasma membrane** → * remains associated as membrane proteins * become part of extracellular matrix * signal other cells

Question 34

Explain the formation of vesicles.

Answer 34

1. **initiation**: coatomer proteins assemble 2. **invagination:** membrane is budding 3. **constriction:** coat proteins shape membrane into a sphere 4. **fission:** vesicle is detached 5. **uncoating:** coat dissasembles

Question 35

Differentiate btw coatomers and for which vesicles they are apparent.

Answer 35

**_clathrin_** * endocytosis * regulated exocytosis: Golgi → plasma membrane * Golgi → late endosome **_COPI_** * retrograde: Golgi → ER * constitutive exocytosis: Golgi → plasma membrane **_COPII_** * anterograde: ER → Golgi

Question 36

Explain the structure of clathrin.

Answer 36

3-limbed structure = **triskelion** each limb: light + heavy chain

Question 37

Explain the process of receptor-mediated endocytosis.

Answer 37

1. assembly of clathrin to cargo receptors on plasma membrane directed by adaptins → **coated pit** 2. **bud formation** 3. dynamin (GTPase) binds → **fission** 4. **vesicle formation** 5. **uncoating** by hsc70 (ATPase)

Question 38

What is the difference btw COPI/clathrin and COPII coated vesicles w/r/t their intracellular transport?

Answer 38

_for COPII:_ 1. **Sar1** binds to plasma membrane 2. **GTP binds** → activates Sar1 → budding initiated ⇒ later: **Sar1 inactivated** when GTP hydrolyzed → coat disassembly _for COPI/clathrin-coated vesicles:_ * **Arf** instead of Sar1

Question 39

What is the function of Rab GTPases?

Answer 39

**_vesicle targeting_** 1. **GEF:** Rab·GDP in cytosol → Rab·GTP 2. Rab·GTP binds to **Rab effector proteins** on vesicle→ tether to membrane 3. fusion w/ membrane → **GTP hydrolyzed,** Rab·GDP released

Question 40

What is the function of SNAREs?

Answer 40

**_initiate fusion_** 1. **v-SNARE** on transport vesicle binds to complementary **t-SNARE** on target membrane 2. **4-helix boundle** 3. ATPase **(NSF) + αSNAP** dissociate 4-helix boundle

Question 41

What is the difference btw antero- and retrograde transport? What is the importance of KDEL receptors?

Answer 41

* **anterograde** = ER → Golgi (COPII) * **retrograde =** Golgi → ER (COPI) BUT: **KDEL** receptors guide vesicles from ER to Golgi → there retained

Question 42

What are G-proteins?

Answer 42

**GTPases** (activated when GTP bound) that transmit signals from **outside stimuli to interior of a cell**

Question 43

What does colchicine do?

Answer 43

_can be gained from autumn crocus_ "mitotic poison" or spindle poison binds to tubulin and **inhibits microtubule polymerization**

Question 44

What are the functions of the cytoskeleton? Differentiate btw filaments.

Answer 44

* **cellular protein scaffolding** → structure and shape * **vesicular transport** and cell division _3 filaments:_ * microfilaments (actin) * intermediate filaments * microtubules

Question 45

Differentiate btw types of actin. Function?

Answer 45

* _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

Question 46

Explain the reassembly of actin filaments

Answer 46

1. _at pointed end of actin filament:_ "old" **actin·ADP** bearing actin depolymerizing factor **ADF** **cofilin** disassembles to actin monomers 2. actin·ADP → **actin·ATP** 3. **profilin** directs actin monomers _to barbed end_

Question 47

Which molecules are responsible for actin branching?

Answer 47

**Arp2/3 complex**

Question 48

Which structures crosslink actin filaments?

Answer 48

Question 49

Explain the structure of a microtubule. Function?

Answer 49

**polymer of tubulin subunits (α + β) → protofilmanets, 13 form→ microtubule** * polarized polymerization (- and + ends) _function:_ * organelle movements * mitotic spindle * cilia and flagella

Question 50

Explain the term dynamic instability w/r/t microtubules. Why is this behavior important?

Answer 50

_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

Question 51

Differentiate btw dyneins. In which direction do dynein and kinesin walk on the microtubule?

Answer 51

cytosolic dynein → **+ end** (axonemal dynein in flagella) kinesin → **- end**

Question 52

What are the properties of intermediate filaments? Classes?

Answer 52

* **no polarity** * **no motor proteins** associated * high tensile strength, resistant to compression, twisting and bending forces * **heterogeneous**