Translocation into chloroplast occurs via a similar strategy
to the one used by mitochondira
Chapter 16-part II
Multiple signals and
pathways target proteins to
submitochondrial
compartments
Both occur post-translationally
Both use two translocation complexes, one at each membrane
Both require energy
Both remove the signal sequence after transfer
However chloroplasts have a H+ gradient across the thylakoid membrane and
use GTP hydrolysis to drive transfer
Matrix targeting
Target:
1.inner-membrane
2.Intermembrane- space
3.Out-membrane: unknow
mechanism
4.matrix
1
Outer-membrane proteins
• Short matrix-targeting sequence is followed by long stretch
of hydrophobic amino acids
Inner-membrane proteins: three separate pathways (B)
Inner-membrane proteins: three separate pathways (A)
Inner membrane has three pathway A,B,C
Inner-membrane proteins: three separate pathways (C)
No N-terminal matrix-targeting sequences
2
Three pathways for targeting inner-membrane proteins
A
B
1. N-terminal matrix
targeting sequence
2. Cleave 1st sequence
3. Hydrophobic stopEnter & redirect
transfer anchor sequence (not clear)
4. Lateral movement
C
1. Multiple internal
sequences
2. Use different inner
membrane translocation
channel
3. Hydrophobic stoptransfer anchor sequence
4. Lateral movement
Oxa1 also participates in the inner-membrane insertion of certain proteins encoded by
mitochodrial DNA synthesized in matrix by mitochondrial ribosomes
Two pathway for transporting proteins from the cytosol to the
mitochondrial intermembrane space (intermembrane-space proteins)
1. Two targeting sequences
2. First N-terminal matrix
targeting sequence removed
3. Second sequence =
hydrophobic stop-transfer
anchor sequence → stay in
membrane
4. Protease cleaves; protein folds
Two pathway for transporting proteins from the cytosol to the
mitochondrial intermembrane space
Intermembrane space
targeting sequence
Direct delivery to the inner
membrane space
3
Outer mitochondrial membrane
Protein target to outer-membrane
Similar to Fig. 16-11
Unclear mechanisms
Need energy
Mitochondrial porin (P70) N-terminal sequence is important
P70 is hydrophobic, for stop-transfer, prevents transfer of the protein
into matrix and anchors protein
Protein target to chloroplast
Targeting of chloroplast stromal
proteins is similar to import of
mitochondrial martix protein
Proteins are targeted to thylakoids by
mechanisms related to translocation
across the bacterial inner
membrnae.
Protein Synthesis in cytosol →
transport → thylkaoid →
photosynthesis
SRP: signal-recognition particle
Have four types of transport protein
to chloroplast: closely related to
bacteria.
Type I: SRP dependent
Type II: related Sec A
Type III: related mitochondrial Oxa 1
Type IV: for metal-containing
protein, ∆pH pathway
R: arginine
Type I
Type IV
4
Type III
Type II
Chloroplast encoded protein
transport into thylakoid
membrane
Fig16-23
Four routes across the thylakoid membrane:
Peroxisome activity:
Peroxisomes are single membrane organelles. Peroxisomes
house a variety lipid oxidation reactions, these often utilize
H2O2 hydrogen peroxide and/or O2 (e.g. catalase & urate
oxidase)
Examples:
B-oxidation & breakdown of fatty
acids
Detoxification of H2O2 (catalase)
2 H2O2
2H2O + O2
Synthesis of certain phospholipids
(e.g. plasmalogen)
Cholesterol breakdown to bile acids
5
16.6 Sorting of peroxisomal proteins
Peroxisomes are bounded by single
membrane.
No DNA and ribosomes, all protein encoded
by nuclear gene.
Has catalase for H2O2 into H2O, are most
abundant in liver cell about 1-2%
Transport protein into peroxisomes by
cytolsolic receptor Pex5 targets protein
with an SKL sequence at the C-terminus
into the peroxisomal matrix.
PTS1: Ser-LyS-Leu (SKL) at C-terminal,
- not cleaved after internalization (very
different)
- translocate folded protein.
Similar to SRP and SRP receptor transport
pathway.
Folded proteins can be translocated across
the membrane
Need ATP.
Synthesis and targeting of perioxisomal proteins
Perioxisomal
targeting sequence
soluble
Encoded by nuclear DNA
Synthesized on free ribosomes
Proteins are folded in the cytosol then transported
Perioxisomal targeting sequence (PTS1)
– Ser-Lys-Leu (SKL)
PST1-tagged protein binds to soluble receptor in the cytosol
Brought to the peroxisomal surface
Moved through translocation channel (not clear)
– Soluble receptor dissociates
– Needs ATP for translocation
– PTS1 is not cleaved
A short signal sequence directs the import of proteins
into peroxisomes
Peroxisomal membrane and matrix proteins are
incorporated by different pathways
Translocation channel protein Pex10,
Pex12 and Pex2 are important.
Any mutation of channel →
catalase can not enter peroxisome
Catalase inner peroxisomes
Pex12
No catalase
Pex19 like receptor for
membrane targeting
Pex3 and Pex16 are
membrane protein for
transport
Division need Pex 11
New peroxisomes are derived by growth and splitting of existing peroxisomes
Fig 16-34 Model of peroxisomal biogenesis and division
6
Zellweger syndrome
Characterized by a variety of neurological, visual, and liver
abnormalities leading to death during early infancy.
Transport of proteins into peroxisomal matrix is impaired;
Genetic analyses of different Zellweger patients & of yeasts
carrying similar mutations identify >20 genes required for
peroxisomal biogenesis.
Zellweger syndrome is caused by the mistakes of
protein import into the peroxisomes.
Accumulation of long chain fatty acids in plasma and
tissues.
Causing severe impairment of many organs and
death.
Peroxisomal disorders affecting either peroxisomal biogenesis or
transport into peroxisomes affect fatty acid and lipid metabolism.
How do proteins get to other organelles?
Mitochondrion
N-terminal
Chloroplast
N-terminal
Peroxisome
C-terminal
Nucleus
Internal
3 – 5 nonconsecutive Arg or Lys residues,
often with Ser and Thr; no Glu or Asp residues
No common sequence motifs; generally rich in Ser, and Thr and small
hydrophobic amino acids, poor in Glu and Asp residues
Usually Ser-Lys-Leu at extreme C-terminus
One cluster of 5 basic amino acids,
or two smaller clusters of basic residues separated by ≈10 amino acids
7
12.3 Macromolecular transport across the nuclear envelop
Nuclear Cargo
Imported
•Polymerases
•Histones
•Transcription factors
•Ribosomal proteins
Exported
•tRNAs
•mRNPs
•Ribosomal subunits
•Transcription factors
106 ribos=>560K ribo proteins imported/min
14,000 ribo subs exported/min
3-4K pores/cell=> 150 ribo proteins/min/pore
Also 100 histones/min/pore etc.
pp.509-514
In nucleus: DNA → premRNA → binding hnRNP
→splicing→m-RNA→
export to cytosol via
nuclear envelop→
translation
From immature to mature
mRNA are associated with
heterogeneous
ribonucleoprotein
particles (hnRNP); mRNA
+ hnRNP → also called
heterogenous nuclear
nuclear RNA (hnRNA)
Mature mRNA + associated
specific hnRNP →
messenger ribonuclear
protein complex; mRNP
Fig 12-1
Nascent transcripts are associated with hnRNP proteins
Protein components with
RNA Recognition Motif
(pre-mRNA)
hnRNPs (heterogeneous ribonucleoprotein particles)
nucleus
cytosol
Nuclear pore complex (NPC)
8
Figure 8.2
Electron Micrograph Showing Nuclear Pores
Figure 8.6
Electron Micrograph of
Nuclear Pore Complexes
eightfold symmetry organized around a large central channel.
Nuclear pore complex (NPC)
Nuclear pore complexes perforate the nuclear
envelope
hydorphilic
Has FG (phenylalanine glycine) amino acids repeat
(hydrophobic)
Also called FG-nucleoporins
< 60kD by water diffusion
Large protein need other carrier or protein participate
Composed by more than
50 different proteins
called nucleoporins.
9
Nuclear pore complex (NPC)
Elaborate (精密) structure of approx. 100 proteins
forming protein lined aqueous channel approx 9nm
diameter
Protein fibrils protrude each side of complex - form
cage-like structure on nuclear side, consist of
nucleoporin (yeast 590 types, mammal 100types)
Typical cell contains 3000-4000 pore complexes
Each pore, on average, imports 100 histone molecules
per minute and exports 6 small ribosomal subunits.
The formed protein also called nucleoporin
Two mechanisms for
nuclear/cytoplasmic transport
• small molecular weight molecules can pass by
diffusion (energy independent)
– < 5000 daltons - freely permeable
– ~17,000 daltons - 2 min equilibration
– > 60,000 daltons- impermeable to nuclear entry.
• But what about ribosomal subunits, mRNP
particles with molecular weights in millions?
Nuclear Pore Complexes (NPC)
• NPCs span the inner and outer nuclear
membrane
– 3000-4000 in typical mammalian cell nucleus.
• NPC are:
– large - 125 million Da
– complex- composed of more than 50 different
proteins
– gated - Have diffusion limit of 50-60 kDa. Larger
proteins require active transport
– busy - every minute each NPC must transport 100
histone proteins, 6 small and large ribosome
subunits, plus numerous other proteins and RNP
complexes.
– traffic is bi-directional and highly regulated.
Transport through NPCs
• Import (chromatin proteins, ribosomal proteins
RNA processing proteins, proteins that shuttle
between nucleus and cytoplasm)
– players:
• nuclear import receptors
• The GTPase Ran and its regulators
• Sequence on protein to be imported -Nuclear localization
signal (NLS)
• Export (ribosome subunits, mRNA complexes,
tRNAs, shuttling proteins )
– players:
• nuclear export receptors
• Ran and its regulators
• Sequence on protein to be exported - Nuclear export
signal (NES)
10
Nucleus imports and exports macromolecules
Nuclear envelope encloses nuclear DNA
Inner membrane contains binding sites for chromosomes and
nuclear lamina
9 nm = diffusion limit
26 nm = active transport
also accumulation versus diffusion
26nm
15nm
9nm
Mechanism of protein import
Outer nuclear membrane resembles ER membrane
Transcription factors enter into nucleus, RNA (once spliced) and
ribosomal subunits are exported out of nucleus
Nuclear envelope perforated by pores, movement occurs in both
directions through these pores
Nuclear localization signals (NLS) direct nuclear proteins
to the nucleus
Proteins selected for import into nucleus have a nuclear
localisation signal (NLS) e.g.
-Pro-Pro-Lys-Lys-Lys-ArgPosition of NLS in protein not important except needs to be on
surface
Protein enter nucleus
nucleus
NLS is recognized by cytosolic nuclear import receptors which
bind to nuclear pore fibrils extending into cytosol
Pore opens and protein plus import receptor enter nucleus.
Import receptor exported for re-use
Protein can not enter nucleus
Keep in cytosol
11
Importins transport protein containing Nuclear-Localizing Signal
into the nucleus
Colloidal gold spheres coated
with peptides containing NLS
Nuclear pore transport
(large aqueous pore) is
fundmental different from
organelle transport
(lipid bilayer).
Cytosol ER → translation to protein→ contain NLS →bind to
importin→ import → nucleus
From digitonin-permeabilized cell system:
Ran (G protein)
NFT2 (nuclear transport factor 2)
importin α and importin β → formed heterodimeric nuclear-import
receptor → α bind to NLS (hydrophobic)of cargo; β bind to FGnucleoprin
Some studies has found only β has import function
Nuclear import: example of a NLS
Simian virus 40 large T antigen (SV40 T)
NLS: PKKKRKV (single amino acid code)
Pro-Lys-Lys-Lys-Arg-Lys-Val
Pyruvate kinase
is cytoplasmic
Digitonin is detergent →
permeabilizes plasma
membrane
Pyruvate kinase +
SV40 T NLS is
nuclear
Synthetic SV40 T-antigen NLS
observation:
Lodish Fig. 11-35
High levels of SV40 T NLS competitively inhibits nuclear import
of NLS-bearing proteins.
Hypothesis:
receptors required for nuclear import.
12
Nuclear import receptors bind nuclear localization signals (α
subunit) and nucleoporins (β subunit)
Nuclear export works like nuclear import, but in reverse
Nuclear export signals & nuclear export receptor & nuclear
transport receptor (karypherins)
tRNA or 5S RNA: nucleiÆ cytosol
NLS-particle: cytosolÆ nuclei
Active nuclear transport is driven
by Ran
• Ran is a small GTPase- binds and hydrolyses guanine
nucleotide triphosphate (GTP)
– Ran exists in two conformational forms: Ran - GTP and RanGDP
– Ran has weak GTPase activity that is stimulated by RanGAP
(GTPase activating protein)
– A guanine nucleotide exchange factor (GEF), called RCC1
stimulates the release of GDP and binding of GTP (higher
concentration of GTP in the cell assures preferential binding of
GTP)
– The cycle of GTP binding, hydrolysis, release provides energy
for nuclear transport
– asymmetric localization of RanGAP and RCC1 assure
directionality of transport
Protein import to nucleus
α+β
Low affinity to NLS
Transport
without ATP
After import to
nucleoplasm →
high
concentration →
export
Interact with
FGnucleoprins
GTPase accelerating protein
Protein import through the nuclear pore complex.
Proteins are transported through the nuclear pore complex in two steps. First the protein
with a classical basic amino acid-rich nuclear localization sequence (NLS) is recognized by
importin α, which forms a complex with importin β. Importin β binds to the cytoplasmic
filaments of the nuclear pore complex, bringing the target protein to the nuclear pore. The
protein and importin α are then translocated through the nuclear pore complex in a second,
energy-requiring step, which requires GTP hydrolysis by the Ran protein.
13
Not all proteins that enter the nucleus have NLS
sequences
Importin beta structure
Nuclear import do not always
bind to nuclear proteins directly.
Soluble
cytosolic
protein
FG-repeat (Phe-Gly) serve as binding sites for the import receptors.
Mechanism of export from nucleus
Most of traffic moving out of nucleus consists of
different types of RNA molecules
RNA moves through nuclear pore as complex of
ribonucleoprotein (RNP)
Protein component of RNP contains a nuclear export
signal (NES) that is recognised by export proteins
Ran = GTPase
GAP = GTPase-activing protein
GEF = Guanine exchange factor
mRNA is bound by hnRNP only after fully spliced so
only mature RNA is exported
14
Heterokaryon assay demonstrating that human hnRNP
A1 protein (red) can cycle in and out of the cytoplasm but
human hnRNP protein C (grn) cannot
HeLa cell + Xenpous
cell → fusion →
heterokaryon (one
cell contain many
nucleus) → add
cycloheximide
inhibited protein
synthesis
Mechanism of transport through nuclear pores
hnRNPC
HeLa cell
Xenpous
hnRNP1
Original: hnRNP only Specific binding
for human HeLa cell
From HeLa hnRNP1 transport to
xenpous nucleus
C: hnRNPA1 protein (nucleus →cytosol → nucleus)
Exportins transport proteins containing NES out of the nucleus
Nucleus export signal
A least three type of NES: PKI (leucine rich), 38-residue in
hnRNP A1,a squence in hnRNP k.
Conformal
change→ high
affinity to
NES
Interact with
FGnucleoprins
hnRNP and mRNA by the same mechanism
Exportin-t : bind to tRNA and interact with Ran → export tRNA to cytoplasm
15
FG-repeat
proteins
Bidirectional model
The Ran GTPase drives directional transport through nuclear pore
complexes
16