Nucleus, Peroxisomes And Mitochondria

Front 1

Organization of the Nucleus

Back 1

1. Nuclear Envelope:
a. inner and outer nuclear membranes
b. perinuclear space
c. nuclear pore complexes
2. Nuclear lamina
3. Nuclear interior:
a. nucleolus
b. nuclear matrix
c. nuclear bodies

Front 2

rRNA and Ribosomal Proteins

Back 2

Large subunit (60S): 50 proteins
- 28S: nucleolus
- 5.8S: nucleolus
- 5S: nucleus

Small subunit (40S): 35 proteins
- 18S:

28S, 5.8S and 18S made from 45S

Front 3

Nuclear Pore Complex

Back 3

- eightfold symmetry - 125MDa
- 30 - 50 different proteins
- cytosolic filaments (import), nuclear basket (export), central gated channel (passive diffusion 8 nm and active transport)

Front 4

Nucleoporin Proteins

Back 4

1. FxF/FxFG (phenylalanine, X, phenylalanine):
- largest
- nuclear import
- filaments, basket, gated channel
2. GLFG (glycine, leucine, phenylalanine, glycine):
- nuclear export
- gated channel
3. No repeats
- located at nuclear envelope
- link NPC to envelope

Front 5

INM proteins
INM, Chromatin, Lamina

Back 5

LAP2: one membrane spanning domain, lamina and chromatin binding domains

LBR: multiple membrane spanning domains, lamina and chromatin binding domains

Front 6

INM proteins
INM, cytoplasm

Back 6

SUN domain on INM proteins
KASH domain on ONM proteins
(actin cytoskeleton)

Front 7

Lamins

Back 7

- intermediate filaments in nucleus
- nuclear localization sequence
- 65kDa and dimers
- head to tail interactions
- phosphorylation - break up, dephosphorylation - regeneration
- during mitosis (prophase): lamins A and C - souble, lamin B - associated with the nuclear membranes

Front 8

Isoprenylation

Back 8

- lamin A and B have CaaX motifs on C terminus
- isoprenylation: farnesyl added by farnesyl transferase at cysteine, membrane association
- first proteolytic cleavage: aaX cleaved off
- carboxymethylation: methyl to C terminus
- second proteolytic cleavage: cysteine and other residues cleaved

both used same protease

Front 9

Lamin A Protein Roles

Back 9

1. nuclear architecture
2. chromatin organization
3. signalling
4. gene regulation

Front 10

Diseases of LMNA

Back 10

- affecting striated muscles, fat tissue, signalling, entire organism
- hutchinson gilford progeria: pre aging disorder - second cleavage prevented and progerin formed (affecting mitosis and signalling)

Front 11

Treatment of Hutchinson Gilford Progeria

Back 11

- FTIs inhibit farnesyl transferase - no progerin formed (geranylgeranyl goup added instead - also very toxic)
- combination of FTI and statin used

Front 12

Diseases of LMNB

Back 12

LMNB1: adult onset, autosomal dominant leukodystrophy
LMNB2: Barraquer-Simons Syndrome - fat loss in upper body and excess fat in the lower body

Front 13

NUPs

Back 13

- NUP358, NUP214: filaments
- NUP62: gated channel
- NUP153: basket

modules that can be formed into NPCs quickly

Front 14

FG Repeats

Back 14

- hydrophobic because of phenylalanine and glycine
- central gated channel
- oily spaghetti model
- reversible hydrophobic interactions with transport receptors

Front 15

Movement across NPC

Back 15

1. Passive diffusion: under 8nm, 40kDa. above 70kDa need a signal
2. Active transport: requires Ran-GTP, transport factors and nups required

Front 16

NLS

Back 16

- not cleaved - permanent
- post translational import
- anywhere in the polypeptide - recognized by receptors (importins)
- positively charged residues (monopartite, bipartite)

Front 17

Importin

Back 17

- two subunits: alpha and beta
β: bind and transport cago themselves or form heterodimers with adaptor α, in which case β binds to FG nups and α binds to NLS

Front 18

NES

Back 18

- hydrophobic amino acids - leucine and isoleucine
- recognized by the CRM1 protein (exportin)

Front 19

Glucocorticoid Receptor

Back 19

- NLS hidden by HSP90
- revealed when glucocorticoid hormone
- changes conformation
- brought into the nucleus - changes gene transcription

Front 20

Importin-α16

Back 20

INM targeting signal - INM proteins stay immobilized by binding to lamina and chromatin
translocon involved

Front 21

Heterokaryon Assay

Back 21

shuttling can be observed
1. cDNA transfected into human cell
2. transfected human cell fused with mouse cell - heterokaryon
3. cycloheximide to prevent protein synthesis

Front 22

Shuttling Proteins

Back 22

- RNA traffic across the nucleus
- NES, NLS, RNA binding domain

Front 23

Polymerases

Back 23

RNA Pol 1: rRNA except 5S rRNA
RNA Pol 2: mRNA hnRNA snRNA
RNA Pol3: 5S rRNA, tRNA

Front 24

TAP

Back 24

help export mRNA out of the nucleus

Front 25

Requirements for mRNA Export

Back 25

1. signal: 5' cap, polyA tail, absence of introns
2. energy
3. transport factors: soluble and nups
4. exporter: TAPs
5. nucleoporins (GLFG repeats)
6. RNA helicases
7. adaptor: REF

Front 26

Export of HIV 1 mRNA

Back 26

1. fully spliced mRNA leaves
2. forms REV protein - contains NES
3. binds to REV response element
4. NES overrides NRS of splicing factors
5. binds CRM1 exportin
6. unspliced, partially spliced mRNA gets out

Front 27

Cellular vs. HIV1 mRNA

Back 27

Helicases:
Normal: Dbp5
HIV: DDX3

Export factors:
Normal: TAP, NXT1
HIV: Crm1

inhibit Crm1 with leptomysin B - not for AIDS since Crm1 needed to transport ribosomes from host

inhibit DDX3 - make sure it isn't similar to Dbp5

Front 28

GLE1

Back 28

- component of RNA export machinery
- trafficking does not work properly if GLE1 is mutated

Front 29

Other Functions of Importin α

Back 29

1. nuclear envelope membrane fusion after mitosis
2. transcription regulation
3. stress response

Front 30

Other Functions of Importin β

Back 30

1. plays a role in mitosis
2. present in stress granules

Front 31

Other Functions of CAS

Back 31

1. exporter for importin α
2. regulates transcription

Front 32

Other Functions of CRM1

Back 32

1. exporter for proteins with hydrophobic nuclear export signals
2. transport through the nucleolus

Front 33

Contents of Stress Granules

Back 33

1. importin α
2. polyA RNA
3. mRNA
4. 40s ribosomal subunits
5. huge list of proteins

Front 34

Catalase

Back 34

enzyme found in peroxisome that removes hydrogen peroxide

Front 35

Peroxisomes

Back 35

- important for detoxification (after medication) removal of H2O2 through catalase
- long fatty acid chain degraded via β oxidation
- synthesis of certain fats - myelin
- diseases: negative effect on nervous system
- bud off the ER
- can continue dividing by fission
- no DNA so all proteins encoded by nuclear genes
- proteins post transcriptionally and can be folded
- require energy in the form of ATP

Front 36

Peroxins

Back 36

encoded by Pex genes and are essential for and mediate peroxisomal biogenesis and protein import

Front 37

Peroxisome Signals

Back 37

1. PTS1: SKL (serine, lysine, leucine) - only at C terminus - predominant signal

2. PTS2: minority signal - mostly at N terminus but also internally - look like mitochondrial targeting signals (mutations) - sometimes but not always cleaved

Front 38

Peroxisomal Import

Back 38

1. protein folded with SKL
2. binds to SKL receptor
3. docks at peroxisomal membrane protein complex with no energy required - 4C
4. translocation using energy and HSP70 in cytoplasm
5. dissociation of receptor and transported back

Front 39

PTS1 (SKL)

Back 39

Pex5p receptor (SKL receptor) - Pex13p, Pex17p

Front 40

PTS2

Back 40

Pex7p receptor - Pex14p

Front 41

Peroxisomal Disorders

Back 41

1. Peroxisome biogenesis disorder:
Zellweger Syndrome - mutation in pex5p - nervous tissue - issues in myelinated neurons - neurological problems

2. Single peroxisomal protein defect:
Hyperoxaluria type 1 - SKL signal becomes a mitochondrial signal

Front 42

Mitochondria

Back 42

- inner membrane, outer membrane, inner membrane space, matrix
- generate ATP
- electrochemical gradient
- HSP60 HSP10 for protein folding

Front 43

Mitochondrial Targeting

Back 43

- signal at the N terminus
- signal is amphipathic helix: positively charged on one side, hydrophobic on the other
- MTS cleaved during transport

Front 44

Mitochondrial Import

Back 44

- protein needs to be unfolded
- cytosolic chaperones keep it unfolded and prevent aggregation by binding to the hydrophobic stretches - require ATP
- binds to receptors on the outer membrane
- moved to a pore where it is translocated
- moved across TOM and TIM (working with PAM) at membrane contact sites
- transmembrane potential needed to get across IM
- HSP70 help pull the protein - motor
- signal peptide cleaved once in the matrix
- HSP70 and HSP60 help refold the protein

Front 45

Targeting to Inner Mitochondrial Membrane

Back 45

1. TOM and TIM: two signal sequences - first one cleaved, second one targets to the IMM through the OXA complex
2. proteins into the intermembrane space through TOM - uses small TIMs and chaperones to insert into IMM - ATP in cytoplasm and electrochemical gradient needed, not HSP70 and 60s

Front 46

Targeting to Outer Mitochondrial Membrane

Back 46

- proteins move across TOM and sometimes into the membrane
- sometimes to the intermembrane space - chaperones and TIMs bind to protein and move to SAM complex
- no electrochemical gradient needed, only ATP

Front 47

Mitochondrial Disorders

Back 47

- Parkinson's
- Human Deafness Dystonia Syndrome - DDP1/TIMM8A mutation
- brain vulnerable since it needs glucose and a lot of energy