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Beyond the central dogma
Central dogma culminates with synthesis of protein in
cytoplasm
But can’t mix proteins, polysaccharides, lipids and nucleotides
together and get a living cell
Formation of a cell requires the context of a pre-existing cell
Cell structures (organelles; mitochondria, chloroplasts, Golgi,
ER) and organization must be inherited, just like DNA
Epigenetics
Lecture 14 cont’d
Intro to protein import into
organelles
Signal sequences
Import into the nucleus
Import into mitochondria and
chloroplasts
Import into ER, vesicle trafficking
Note - in the next few lectures I will show many figures
from Molecular Biology of the Cell 4th ed. (Alberts et al.)
On reserve at Marriott
Nuclear import occurs through pores in the double membrane
“Nuclear envelope”=
Outer nuclear membrane
ECB 15-7
Inner nuclear membrane
Perinuclear space
ER
Nuclear pores
All nuclear transport occurs through
nuclear pores
What molecules must be imported into nucleus?
Exported?
Nuclear lamina
Nuclear pores are large protein complexes
Cytplasmic
fibrils
Cytosol
Cytoplasmic face
Nuclear envelope
Nucleus
Nuclear lamina
Annular subunit of central
channel or transporter
Nuclear basket or cage
Nuclear face
ECB 15-8
Multiple copies of ~100 different proteins (nuclear
pore proteins = NPPs) totaling >125 million daltons!
Transport of large molecules is active requires GTP
Small molecules (< 60
kDa), or about 9 nm
diameter) enter or
exit nucleus by
passive diffusion
Nuclear pores also required for active export of RNPs
(including ribosome subunits, mRNA, tRNA etc.)
Larger molecules
must be actively
tranported:
(1)
binding to
transporter;
and
(2) transport thru
nuclear pore
using GTP
Import and export occur through same pores
A nuclear localization signal (NLS) is necessary and sufficient
for nuclear import of proteins
The “classical” signal for nuclear import includes multiple basic amino acids
(K = lysine and R = arginine)…example P-P-K-K-K-R-K-V
NLS can be anywhere in protein sequence
Simplified view of nuclear transport
NLS
(cargo)
(importin)
Pore opens
ECB 15-9
Energy for transport provided by G proteins
(GTP binding proteins; large family)
Molecular “switches”
Pi
GAP
GDP
GTP
GTPase
“on”
GTP
GEF
GTPase
“off”
GDP
GAP = GTPase Activating Protein
GEF = Guanine Nucleotide Exchange Factor
Pi
RAN
GTPase used in
nuclear transport
GAP
GDP
GTP
GTPase
“on”
GTP
GEF
GTPase
“off”
GDP
Nuclear import/export cycle is driven by GTP hydrolysis
Q: relation to protein
transport??
Directional protein import is driven by GTP hydrolysis
NLS
Importin
Importin
Pi
+
NLS
Ran
GAP
Cytoplasm
Ran-GTP
Importin
Ran-GDP
Importin (NLS receptor) binds
cargo (with NLS) in cytoplasm
Importin-cargo transported into
nucleus thru nuclear pore
Ran-GTP in nucleus binds importin,
importin releases NLS (cargo)
Ran-GTP-importin exported from
nucleus thru pore
Ran-GAP stimulates GTP
hydrolysis in cytoplasm by Ran
Ran-GDP releases importin in
cytoplasm
Nucleus
NLS
Importin
Importin
Ran-GTP
GTP GDP
Ran-GTP
Ran-GDP
RanGEF
NLS
Ran-GDP transported into nucleus
(not shown)
Ran GEF stimulates nucleotide
exchange restoring Ran-GTP.
Specific signals direct export from the nucleus: lessons from HIV
Human immunodeficiency virus
(HIV) is a “retrovirus:”
Human T lymphocyte
Transport
DS vDNA
RNA genome with DS DNA
intermediate
Reverse
transcription
vRNA
Integration
Uncoating
RNA is “reverse transcribed” to
make DS DNA
Unspliced vRNA is trapped in
nucleus (contains introns-no export)
Transcription
Unspliced vRNA (9 kB)
HIV
Processing
GpppC
mRNA (2 kB)
Progeny virus exits
host cell by budding
Alternative splicing produces over
30 mature mRNA that are
exported and translated
AAA
Nucleus
Rev
Transport
Translation
Cytoplasm
Nuclear Export Signal Rev is req’d for export of RevvRNA from nucleus
One protein, Rev contains a NLS
and is tranported into the nucleus
Rev binds “Rev-response element”
on vRNA
Rev-RNA complex exported, RNA
packaged and virus leaves cell
Lecture 14
Intro to protein import into organelles
Import into the nucleus
Import into mitochondria and chloroplasts
Recall mitochondrial and chloroplast structure
Organelle DNA - encodes small % proteins, human mitochondria encode
only 13 proteins
Rest (thousands) encoded in nucleus, transcribed, exported to
cytoplasm, translated and imported into correct organelle
And correct compartment in that organelle
Import into mitochondria is post-translational
Translate mRNA for
mitochondrial matrix
protein in vitro
Proteins contain Nterminal “signal
sequence”
Trypsin
Add “energized”
mitochondria
Protein
imported into
mitochondria
Trypsin
Imported matrix protein is protected from
added protease
Digest with protease
Protein degraded
Import into mitochondria and chloroplasts is
post-translational
Import is directed by a signal sequence at the Nterminus of mitochondrial proteins
Non-polar aa (green) on the other…
Positive charge (red) clustered on
one face of helix…
No conserved sequence
Predicted to form “amphipathic” a-helix
Cleaved after protein is imported
MBoC (4) figure 12-23
© Garland Publishing
TOMs and TIMs: Import into the mitochondria
matrix requires two membrane transporters…
Transport thru aqueous channels: “TOM” and “TIMs” (Translocaters in
Outer/Inner Membrane)
Mitochondrial import signal binds receptor in outer membrane (assoc w “TOM”)
“Contact site” (close
apposition of OM & IM)
Cytoplasm
Outer membrane
Intermembrane space
Inner membrane
“TIM23”
Matrix protein w
N-terminal signal
sequence
Import
receptor
“TOM”
Removal of
signal sequence
Matrix
Mature
matrix
protein
See ECB figure 15-10
Protein import into mitochondria requires energy…
Cytosolic
HSP70
ADP + Pi
ATP
Cytoplasm
Outer membrane
TOM
IMS
++++++
Inner membrane
Matrix
----
+++++++++
TIM23
----------
Adapted from MBoC (4) figure 12-27
© Garland Publishing
ATP
Mitochondrial
HSP70
ADP + Pi
(1) Electrical potential (DY) across inner membrane req’d to initiate transport
(2) Cytosolic HSP70 unfolds protein for import (ATP used)
(3) Mitochondrial HSP70 refolds protein after import (ATP used)
How are proteins targeted to other mito membranes/compartments?
../L14OrganelleImport/15.5-mito_import.mov
How are proteins targeted to mitochondrial membranes and
compartments? …the direct route
Cytoplasm
TOM
Protein in IM
Protein in IMS
Outer membrane
IMS
Inner membrane
TIM
Matrix
“Stop transfer” signal
Cleaved stop transfer
(degraded)
Matrix signal
(cleaved and degraded)
Adapted from MBoC (4) figure 12-29
As before, signal sequence directs import through TOM/TIM23…
“Stop transfer” signal interrupts translocation through TIM23, releasing
protein to inner membrane…
Cleavage of stop transfer signal releases protein to intermembrane space…
Import into the thylakoid requires multiple signals
Transporters
Cytosol
Outer membrane
Transit
peptide
IMS
Inner membrane
Receptor
Transit peptide cleaved
Stroma
Thylakoid
signal
Thylakoid
A “transit peptide” (an amphipathic helix) targets to chloroplast stroma
(similar to mitochondrial signal peptide, but NOT interchangeable!)
Evidence for four paths to thylakoid
Adapted from MBoC (4) figure 12-30 © Garland Publishing
Protein targeting
NLS: (basic)
Protein
targeting
Nucleus
NES: (L-rich)
Mitochondria
Signal
peptide
Cytoplasm
Additional signals for
subcompartments
Chloroplasts
Vesicle targeting
RER
See ECB figure 15-5
Golgi
Endosomes
Secretory
vesicles
Plasma membrane
Transport
Retrieval
Next two lectures
Lysosomes
L15: Protein and vesicle targeting
Today - import into
ER, begin vesicle
targeting
ECB 15-5
ER network is extensive
GFP-protein in plant cell ER
TEM of RER in dog pancreas
Note ribosomes on membrane
Vesicles derived from ER by biochemical prep are termed microsomes
Some ribosomes bind to ER
ECB 15-12
What is the evidence for cotranslational transport?
Transport of protein into ER is cotranslational
Translate mRNA in vitro…
In vitro product ~2 kDa larger than in vivo
product
~15-25 addtnl aa at N-terminus
Add protease - product degraded
Add RER microsomes
AFTER translation…
Product still ~2kDa larger than
in vivo product…
Add protease…
Product degraded…
Add RER microsomes
DURING translation…
Product processed to mature form…
Add protease…
Product protected…
INSIDE microsomes!
The “Signal Hypothesis”
From results of experiments such as these, Dobberstein
and Blobel proposed a hypothesis
1. The signal for translocation of a secretory protein into
the ER resides in the nascent polypeptide, in the form of
a leader “pre-” sequence or “signal peptide;”
2. Translocation of the polypeptide across the ER
membrane is co-translational (unlike import into nucleus,
mito, and chl); and
3. the signal peptide is cleaved post-translationally in the
ER lumen by a “signal peptidase.”
Blobel - Nobel prize 1999
ER Signal Sequence
No conserved sequence
Signal sequence is 12-25 amino acids
Predicted to form a-helix with
hydrophobic core (yellow aa above)
Signal sequence is both necessary and sufficient for
import into ER
Necessary
Sufficient
Requirements for targeting and translocation into the ER
1.
“Signal sequence” : hydrophobic a-helix in nascent protein
2. “Signal recognition particle (SRP):” cytoplasmic complex of
protein and RNA binds signal sequence
3. “SRP-receptor:” integral ER membrane protein
4. “Translocon:” an aqueous channel through ER membrane
(sec61 complex)
Targeting to RER
ECB 15-13
1.
Translation exposes signal sequence outside ribosome
2.
SRP -a complex of 300bp RNA and 6 proteins- binds the signal sequence in
nascent protein, transiently arrests translation
3.
SRP-arrested ribosome binds SRP receptor in ER membrane (targeting)
4.
Ribosome and polypeptide handed to a translocation channel (“translocon”). SRP
and SRP-R are recycled (requires GTP hydrolysis). Translation resumes and
translocation begins
Proteins destined for secretion enter ER lumen
ECB 15-14
Signal peptide targets nascent protein to RER as before
Signal peptide is cleaved by signal peptidase associated with translocation channel
Translation and translocation are completed, releasing completed polypeptide into
lumen of RER
Signal peptide is degraded
What about membrane proteins??
Membrane proteins contain stop transfer sequence
ECB 15-15
As before, signal peptide targets nascent protein to RER
However, “Stop transfer” sequence halts translocation
Protein is released from translocon
Stop transfer sequence acts as transmembrane domain
Double- and multipass membrane proteins
ECB 15-16
Internal signal sequence targets nascent protein to RER…
“Stop transfer” sequence halts translocation and releases protein from translocon…
Signal sequence and stop transfer sequence act as transmembrane domains
Protein folding in the ER is assisted by “BiP”…
“Binding protein” (HSP70 family of ATPases) in ER lumen binds nascent
polypeptide as it is being translocated, and assists folding (and translocation?)…
C
N
Signal peptide
Signal
peptide
N
RER membrane
ER Lumen
Translocon
(sec 61 complex)
Adapted from MBoC (4) figure 12-46.
See ECB figure 15-14
BiP
ADP+Pi
Signal
peptidase
ATP
BiP
BiP binds nascent protein during translation/translocation…
C
N
“Secreted
protein” in
lumen of RER
Release of BiP from folded polypeptide requires energy (ATP)…
Incorrectly folded proteins are held in ER until folded properly, or are targeted
for degradation…
The topology of a membrane protein can be predicted…
Hydropathy plot for Rhodopsin
1
2
3
4
5
6
Topology of Rhodopsin
7
COOH
Hydrophobic
A
B
C
D
Cytoplasm
1
2
3
4
5
6
7
Hydrophilic
ER Lumen
NH2
Adapted from MBoC (4) Figure 12-50 © Garland Publishing
Hydrophobic a-helices of 15-25 aa are predicted to be membrane spanning
domains…and also function as “topogenic sequences.” Seven domains in rhodopsin
100
200
“Start transfer” initiate protein translocation, “Stop transfer” sequences halt
translocation…
Note start sequences can be in either orientation
H2N-
1
Start
A
2
3
Start
Stop
B
4
5
Start
Stop
C
6
7
Start
Stop
D
-COOH
Review of the “Signal Hypothesis”
1.
The signal for translocation/insertion of a protein into the ER membrane
resides in the nascent polypeptide, in the form of a “signal sequence.”
2.
Translocation of the polypeptide across the ER membrane is co-translational
3.
The signal peptide (of secreted proteins) is cleaved post-translationally in
the ER lumen by a “signal peptidase.”
4.
Four components: (1) signal sequence, (2) SRP, (3) SRP-R, and (4) translocon
5.
Uncleaved signal sequences (and “stop transfer” sequences) function as
transmembrane domains in integral membrane proteins…
6.
The topology of a protein can be predicted from the “hydropathy” plot of its
amino acid sequence…
15.7-ERprotein_trans.mov
Vesicle targeting
Protein and vesicle targeting
NLS: (basic)
Protein
targeting
Nucleus
NES: (L-rich)
Mitochondria
Signal
peptide
Cytoplasm
Additional signals for
subcompartments…
Chloroplasts
Vesicle targeting
RER
See ECB figure 15-5
Golgi
Endosomes
Secretory
vesicles
Lysosomes
Plasma membrane
Transport
Retrieval
15.1-cell_compartments.mov
Membrane cycling
endocytosis
ECB15-17
Exocytosis
(secretion)
Secreted proteins
Plasma membrane
proteins
Transport is highly regulated so vesicles carry appropriate cargo for their
specific destination
Lumen of organelle is equivalent to outside of cell
x
x
x
Begin with ER to Golgi
x
x
x
What about membrane protein in ER?
Modification of proteins begins in ER
Disulfide bridges
Glycosylation -
ECB 15-22
common in plasma
membrane and
secreted proteins
Asn-X-Ser
Most common glycosylation is addition of a specific oligosaccharide (14mer) to
asparagine during translation. Addition is to the NH2 group; N-linked glycoproteins
Addition is done in a single step by transfer from specialized dolichol lipid
This oligo is then extensively modified in diverse ways
Modification begins in ER: Transported to Golgi for more processing
From the ER, proteins are transported to the Golgi
Nuclear envelope
RER
Vesicular-tubular
clusters to CGN
Golgi
MBoC (4) figure 13-22 © Garland Publishing
Proteins leave the ER in transport vesicles budding from exit sites…
Transport vesicles from ER fuse to form vesicular-tubular clusters…
Vesicular-tubular clusters enter the Golgi by fusing with the cis-Golgi
network (CGN)
Glycoproteins are “processed” as they pass thru the Golgi…
From the ER, proteins are transported to the Golgi
cis-Golgi
network (CGN)
Vesicular-tubular clusters in from RER…
cis
medial
trans
Trans Golgi network (TGN)
ECB 15-24
Proteins leave the ER in transport vesicles budding from exit sites…
Transport vesicles from ER fuse to form vesicular-tubular clusters…
Vesicular-tubular clusters enter the Golgi by fusing with the cis-Golgi
network (CGN)…
Glycoproteins are “processed” as they pass thru the Golgi…
The Golgi is biochemically compartmentalized…
Nucleotide diphosphatase (trans)
Osmium (cis)
MBoC (4) figure 13-28
© Garland Publishing
Acid phosphatase (TGN)
Glycoproteins are further processed in the Golgi
Protein synthesis
ER
Golgi apparatus
CGN
cis
Removal of mannose
Addition of GlcNAc
Glycosylation at
H3N+…XXNXSXX…COOAs protein moves through
Golgi, monosaccharides are
added or removed in specific
Golgi compartments
medial
GlcNAc = N-acetylglucosamine
Mannose
Glucose
Addition of galactose
trans
TGN
Lysosome
Plasma membrane
Constituitive
secretion
(Default?)
Regulated
secretion
Secretory
vesicles
Fucose
Galactose, etc.
Proteins are sorted
in the TGN…
Constitutive secretion…
Regulated secretion…
Lysosome…
Why are membrane/secreted proteins glycosylated?
Structure and folding?
Protection of cell
(protein) from external
proteases?
Function?
adhesion…
signaling…
The plasma membrane of many (most?) cells is
coated with glycoproteins
Transport through Golgi
cis-Golgi
network (CGN)
Vesicular-tubular clusters in from RER…
1. Vesicle transport
2. “Cisternal
maturation”
“Budding”
cis
Transport
vesicles
“Fusion”
medial
trans
trans-Golgi
network (TGN)
ECB 15-24
Transport vesicles out
Cisternal maturation and vesicle transport probably both
contribute to membrane flow through Golgi
Next time
Vesicle transport from ER to Golgi
Transport from Golgi
Constitutive secretion
Regulated secretion
To lysosome