Organelles And Cell Signaling

Front 1

What is the role of the nucleolus in the biogenesis of the ribosomal machinery?

Back 1

NCL1 and NPM1 bind pre-rRNA

Front 2

Analyze this data.

Back 2

-Triton causes membrane to vesiculate
>causes transmembrane proteins to be released into supernatant (integral components)
-NaOH causes peripheral proteins to be released into supernatant
-huge detergeant resistant structure of RBC membrane

Front 3

What are these 2 graphs showing?

Back 3

-2 patterns of protein uptake in cells
>A=non-specific
>B=receptor-mediatedlevel-off due to receptor saturation

Front 4

Describe the endocytosis of EGFr.

Back 4

-EGF= Y kinase (autophosphorylated)

Front 5

Describe how the clathrin coat is recruited.

Back 5

-intracellular C-terminal tail has signaling peptide recognized by apdaptin (brings in clathrin coat)

Front 6

What is responsible for pinching off the clathrin-coated vesicle?

Back 6

-dynamin=G protein; allows budding of vesicles
-gold particled dynamin EM micrograph

Front 7

How is a receptor targeted for clathrin-mediated endocytosis?

Back 7

-ubiquitin recognized by UIM-adaptor
-ubi=8 kD protein added to substrate to target for degradation (usually)
>E1, 2, and 3 are enzymes needed to ubiquitinate
>Cbl puts ubi on
>Nedd4=HECT E3
>UBP=de-ubiquitinater
>UIM addapter removed by its own ubi
(these proteins are part of adaptin complex

Front 8

What are 3 fates of bound ligand?

Back 8

-polarized epi cell
>2 PM domains
>recycling, degredation, and transcytosis are 3 options for a bound ligand

Front 9

Interpret this data.
(transferrin on left, EGFr on right)

Back 9

-fluorescently labeled protein added to cell to visualize localization
>transferrin disappears more quickly than EGF
>this is b/c transferrin is recycled (after Fe release) whereas EGF is degraded

Front 10

Describe the uptake of iron into a cell.

Back 10

-transferrin maintains Fe conc. In cells
3) uncoating clathrin and adapters recycled back to surface
4) Late endosomes are acidic
>apotransferrin binds receptor under acidic conditions whereas transferrin only binds at neutral pH
5) Apotransferrin released at neutral pH
**neither transferrin nor receptor are degraded**
-transporter in endosome shuttles Fe to cytosol

Front 11

What dictates the destination of vesicles?

Back 11

-Rab proteins keep endosomes going on a specific path
-each Rab has an effector
>disctates fusion and docking of vesicles

Front 12

What components tether a vesicle to a membrane?

Back 12

Front 13

What components are involved in membrane fusion?

Back 13

-late and early endosomes tend to fuse
-late endosomes and lysosomes tend to fuse

Front 14

What are 3 fates of endocytosed receptors?

Back 14

Proteins that are internalized by receptor-mediated endocytosis may be either transcytosed, recycled, or degraded with or without their receptors.

Those ligands that are recycled (transferrin) transit only the early endosome while ligands (EGF and LDL) destined for lysosomal degradation transit both early and late endosomes.

Front 15

What components are required for vesicular trafficking?

Back 15

Vesicular trafficking requires GTP, cytosolic GTP-binding proteins (RAB’s), cytosolic SNAP’s, NSF and membrane-bound SNARE’s.

Front 16

Describe the late endosome.

Back 16

-late endosome tends to target for degradation
-recycling usually occurs in early endosome
-Y kinase of EGFr is cytoplasmic in late endosome
>in multivesicular body, kinase aspect is internalized

Front 17

What factors are needed for the invagination of the late endosome for receptor degradation?

Back 17

-ESCRT complex-mediated budding of multi-vesicular body
>doesn’t utilize clathrin or calveolin
>Ubi stil required (mono)

Front 18

How does endocytosis play a role in thyroid metabolism?

Back 18

-thyroxine synthesized as thyroglobulin
>Y moieties of thyroglobulin cross-linked at apical surface, then stored in colloid
>Y iodonated also
>cell endocytoses colloid and degrades everything but T3 and T4

Front 19

How does endocytosis play a role in cholesterol metabolism?

Back 19

-LDL disocciates from receptor in late endosome due to acidity
>cholesterol released for membrane integrity

Front 20

What is the basis of familial hypercholesterolemia?

Back 20

-hypercholesterolemia (familial) all based on receptor problems
B) 4 aa seq missing from LDLr
>no apater recognition=no endocytosis

Front 21

Interpret this data.

Back 21

b) At 4C, just see binding b/c there’s no internalization or degradation at this temp (shows receptor #)
>at 15C, internalization doesn’t reach lysosomes

Front 22

Contrast how endocytosis fx the different components covered in this lecture.

Back 22

Endocytosis down regulates tyrosine kinase receptors, up regulates thyroxine production, and modulates the intracellular transport of needed iron and cholesterol.

Front 23

What are the lysosomal functions?

Back 23

Degradation of endogenous and exogenous macromolecules and recycling of monomeric subunits (e.g., amino acids)

Degradation of aged or damaged organelles

Removal of intracellular pathogens

Homeostasis of iron and cholesterol

Antigen presentation (e.g., macrophages)

Bone remodeling (e.g., osteoclasts)

Cell survival and Cell death

Front 24

How is lysosomal pH determined?

Back 24

Determinants of lysosomal pH. Lysosomal acidification is dependent on V-ATPase, a large multimeric enzyme complex that transforms the energy of ATP hydrolysis into the movement of protons across the lysosome membrane. Electrogenic proton transport creates an electrical gradient that must be dissipated to establish the substantial chemical proton gradient. Electroneutrality can be maintained through the parallel influx of anions alongside protons. ClC-7, a chloride proton antiporter, and CFTR have been proposed to constitute the counter ion pathways in the lysosome membrane, as described in the text. The efflux of cations (C+) through distinct channels or transporters can also occur. Parallel proton leak pathways (dotted lines) are also known to exist and require continued V-ATPase activity to maintain a steady-state pH. Acidification kinetics are also contingent on the luminal buffering power (not depicted).
-V-ATPase is similar to F1/F0 ATPase of mitochondria
>pump requires ATP
>other ion channels modulate pH
-many membrane proteins of lysosomes have COOH moieties that become salicylic acids that prevent the protein from being degraded by hydrolytic enzymes

Front 25

Compare/contrast protein vs. carbohydrate degradation in the lysosome.

Back 25

-carbs synthesized and degraded in a step-wise manner
>lots of redundancy in protein degradation, but not carbo degradation
>many disorders of carbo deg.
-endopeptidases cleave protein seq in middle
-exopeptidases cleave from N- or C-terminus (1 or 2 aa’s only)

Front 26

Analyze this data.
(Hydroxychloroquine added in panels on right)

Back 26

Hydroxychloroquine induces lysosomal membrane permeability (lmp).
-cathepsin beta=soluble, hydrolytic proteinase
>prob not active at neutral pH
>leaks out when treated with hydroxychloroquine (anti-malarial and anti-cancer Rx)
-LAMP1=membrane protein (i.e. membrane still in tact)

Front 27

Describe lysosomal biogenesis.

Back 27

Lysosomal proteins are synthesized on the rough endoplasmic reticulum and post-translationally modified in the RER and Golgi apparatus.

Lysosomal proteins are sorted in the Golgi apparatus to the late endosomes and lysosomes. This sorting requires AP-1 or AP-3

Soluble enzymes have a mannose 6-phosphate signal.

Membrane proteins have a peptide signal.

Front 28

Describe the trafficking of lysosomal enzymes.

Back 28

the M6P residues that direct proteins to lysosomes are generated in the cis-golgi by 2 resident enzymes:
1) N-acetyglucosamine (GlcNAc) phophotransferase transfers a phospho-GlcNAc to 1 or more mannose residues (added in ER). Only lysosomal enzymes have seq that are recognized and bound by this enzyme, so phospho-GlcNAc gp's are added only to lyso enzymes
2) After release from phosphotransferase, a phosphodiesterase removes GlcNAc gp, leaving phospho-mannose on lyso enzyme

Front 29

Analyze this data.

Back 29

-mucolipidosismiss GlcNAC phosphotransferase enzyme (GNPtab)
>looking at exocrine glands
>LAMP2=lysosomal protein
-the soluble enzymes normally within the lysosomes are missing, and thus, fatty droplets accumulate and cause the vesicles to swell

Front 30

Compare the differences of presentation in an acinar/exocrine cell vs. a fibroblast that has lost Gnptab (GlcNAc) activity.

Back 30

-exocrine cells have regulated secretion.
>they are normally secreters so to become constitutively active in the secretive pathway doesn't impact as much as fibroblasts which usually retain more of the enzyme

Front 31

What are the various delivery pathways to lysosomes?

Back 31

Phagocytosis of extracellular organisms such as bacteria and yeast

Receptor-mediated endocytosis of extracellular hormones, proteins, macromolecules, lipid particles, and viruses

Pinocytosis of extracellular proteins and glycosaminoglycans

Crinophagy of intracellular secretory vesicles

Chaperone-mediated transport of endogenous soluble proteins

Microautophagy of endogenous soluble proteins, ribosomes, and glycogen

Macroautophagy of macromolecules and organelles

Front 32

Describe the merger of the endocytic and biosynthetic pathways.

Back 32

Front 33

Compare micro- and macroautophagy.

Back 33

-microautophagy= invagination of lysosome (direct route)
-macroautophagy=indirect route requiring fusion event

Front 34

What chaperone mediates selective transport of cytosolic proteins to the lysosome for degradation?

Back 34

There exists a chaperone (hsc73) that mediates selective transport of cytosolic proteins into the lysosomes for degradation.

Lysosomal transport requires ATP, lysosomal membrane protein (LAMP2a), a hsc73 recognition domain within the target protein (KFERQ), and cytosolic and lysosomal hsc73.

Front 35

What are some properties of hsc73?

Back 35

-soluble protein that traverses lysosomal membrane
>protein stretched to fit through channel
>recognize KFERQ signal

Front 36

What is LAMP2? What disease does a defect cause?

Back 36

-lysosomal membrane protein
-The results suggest that Lamp-2 is required for both chaperone-mediated autophagy and macroautophagy.
-Danon disease is an X-linked disorder in which cells have a defective Lamp-2 This disease is clinically characterized by cardiomyopathy, myopathy, and mental retardation.

Front 37

Describe the breakdown and reformation of the nuclear envelope during mitosis.

Back 37

Front 38

Describe lamins and their function(s).

Back 38

-lamins are class V IF's
>chromatin may attach directly or connect via other adapters

Front 39

Describe the structure and processing of lamins.

Back 39

-farnesyl residue inserted at Cys of pre-lamin
-splicing leads to B1 and B2
-NLS, IgG repeats, CAAX box
-FACE1 cleaves CAAX box to yield mature lamin
-pre-lamin A, 646 recognized by FACE1 to cleave CAAX
>mutation makes FACE1 unable to work
>Hutchinson-Gilford progeria syndrome (G608)

Front 40

How are large proteins (>9 nm/60 kD) transported across the nucleus?

Back 40

-sometimes requires adapter protein

Front 41

Describe the mechanism of nuclear import.

Back 41

-mechanism of nuclear import:
>4 components: RAN (GTP or GDP), cargo, importin (receptor)
-RCC!=Ran GEF
>balance of GEF and GAP in cytoplasm vs. nucleoplasm drives import of materials
(protein maintains tertiary structure...NPC accomodates protein shape)

Front 42

Describe export through the NPC.

Back 42

-must have export signal to be shuttled out of nucleus
>reverse process of import

Front 43

Describe Ran-independent export of mRNA's.

Back 43

-Tap and Nxt1 recognize mature mRNA (binds close to polyA tail)
-Dbp5=RNA helicase
>provides ATP to move from nucleus to cytoplasm

Front 44

What are the functions of the peroxisome?

Back 44

Front 45

Describe peroxisome biogenesis.

Back 45

Pex 19=peroxisomal receptor
>facilitates association of other membrane proteins
-Pex 5 recognize cargo protein with PTS-1, Pex7 recognizes PTS-2
>comprise mature peroxisome (with other factors) to recruit needed enzymes to core
-mature peroxisome duplicates through fission using Pex11

Front 46

Give an overview of the peroxisomal matrix protein import cascade.

Back 46

Front 47

What are the key players in the peroxisomal matrix protein import cascade.

Back 47

i)Cargo recognition in the cytosol and direction of the receptor-cargo complexes to the peroxisomal mem:
>2 major targeting signal Pex5p (perox. targeting signal 1 receptor) and PTSR2 have been id'd
>The Pex5p signal is a carboxy-terminal tripep S-K-L-COO-
>pro harboring the PTS1 signals are recognized by the soluble receptor Pex5p (PTSR1)
>PTS2 signal pro's are recognized by Pex7p (PTSR2)
ii)translocation of the receptor-cargo complex to the luminal site of the peroxisomal mem:
>the receptor-cargo complexes assoc with the peroxi mem via the peroxi docking complex (Pex14p, 13p, and 17p)
>the RING-finger domain containing peroxins (pex2p, 10p, and 12p) form the RING-finger complex, which is connected to the docking-complex via Pex8p.
iii)disassembly of the receptor-cargo complex in the peroxi lumen:
>receptor-cargo complex dissociates at the luminal site of the mem
>integral PTSR1 is either monoubi by the E2 enzyme Pex4p or polyubi by Ubc4p or Ubc5p
iv)return of the receptor to the cytosol:
>the ATPase assoc with various cellular activities (AAA) peroxins Pex1p and 6p, which are anchored to the peroxi mem by Pex15p, dislocate the ubi Pex5p from the mem back to the cytosol
>polyubi PTSR1 are degraded by the proteasome, whereas monoubi receptors are recycled for futher rounds of import
(-import cascade:
>Pex5 and 7 recognize cargo based on short seq
>Pex 7 assoc w/Pex 18 and 21 to form complex
>translocates to membrane andis recognized by ring-like structure composed of different Pex's (13, 14, and 17)
>dissociates once inside
>Pex 5 and 7 get exported back out
>if mono-Ub, get moved out by Pex1p and 6p
if poly-Ub=proteasomal degradative pathway)

Front 48

Describe what a mutation in Pex12 or 3 would look like.

Back 48

-mutations involved in biogenesis or transport drastically effect the cell
-Pex12 mutation blocks import
-Pex3 mutant blocks biogenesis
>don't get inserted in membrane (remain diffusely in nucleus)

Front 49

What are the 2 types of peroxisomal diseases?

Back 49

Front 50

Describe the mitochondrial fusion proteins.

Back 50

-all 3 fusion proteins are GTPase's, with TM domains and HR domains
>TM anchors to outer mito. membrane
>HR anchors HR2 from neighboring mito. and pulls the 2 together
OPA1 within the mito. becomes active once the 2 mito. are connected and allows fusion

Front 51

Describe the mitochondrial fusion process.

Back 51

Front 52

Describe mitochondrial fission proteins.

Back 52

Front 53

Describe mitochondrial fission.

Back 53

Initial constriction of fission process is self-directed after maturation
>Drp1 recognizes this groove
-once seqperated, 1 mito. has high nrg capacity and the other is low nrg
>low nrg gets degraded

Front 54

Compare FA oxidation in the 2 organelles in which it occurs.

Back 54

-acetyl CoA produced in FA oxidation in both peroxisomes and mito.
>acteyl CoAmust be transported from peroxisome to other organellles
-e- transport system necessary for reducing O2 to water

Front 55

Describe the process of mitochondrial oxidation damage.

Back 55

Front 56

Describe the role of SOD in human disease.

Back 56

Front 57

What are some ways SOD mutants are implicated in motoneuron diseases?

Back 57

mutations in SOD lead to aggregate formation (vs. SOD homodimers)
>aggregates complex with Bcl2 in mito. mem.
>can cause mito. swelling, affect permeability, or release CytC (apoptotic)
>can be tissue specific (may effect neuronal system more so than other organs)

Front 58

What organelle is often targeted by SOD1?

Back 58

xs e- gradient produced by stroke (due to blockage of ATP production)
>produce ROS
>p53 involved in trancscription of mitochondrial protein
>when activated, can bind to mito. mem. and transcribe

Front 59

How do ROS initiate apoptosis?

Back 59

-mito-specific genes
>binds Bcl protein (in complex w/BAX)
>BAX gets released (also, actively being transcribed
>xs BAX forms a pore in mito. mem. though which Cyt C gets released and initiates apoptosis

Front 60

Compare peroxisomes and mitochondria.

Back 60

Front 61

Describe the role of SOD in ALS.

Back 61