bacterial cellweb.mit.edu/7.01x/7.012/psets/pdfs/f04_specific/10-13_handout.pdf · outer membrane...
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Cytoplasmchromosome
PeriplasmInner membrane
Outer membrane
Outside the cell
BACTERIAL CELL
mRNA
protein
EUKARYOTIC CELL
Mitochondrion
Plasma membrane
Nucleus
Endoplasmic Recticulum Golgi
Apparatus
Cytoplasm
Outside the Cell
N
EukaryoticCell
Bacteria
InsulinGrowth Factors
Antibodies
Insulin ReceptorGrowth Factor
Receptors
Histidine synthesisLactase
Glycolysis EnzymesCyclins
ToxinLactose Receptorβ−galactosidase
FullySecretedProtein
(Outside the Cell)
MembraneProtein
Cytoplasmic Protein
Examples
George Palade
Hamster pancreatic cellNucleus
Mitochondrion
Earliest Time point
Next observed location
LocationAfter Golgi
Millstein
“in vitro synthesis of immunoglobulin light chains. …To our delight we ran into the unexpected observation
of the existence of a biosynthetic precursor oflight chains. Further experiments led us to propose
the extra N-terminal sequence was a signal forvectorial transport across the membrane during
protein synthesis. That was the first evidence whichindicated that the signal for secretion was an N-terminal
segment, rapidly cleaved during protein synthesis.”
FROM NOBEL LECTURE 1984
CytoplasmicExtracts
N
Messenger RNARibosomes &
charged tRNAs
Microsomes(RER vesicles)
in vitroBlobel
+added late
+
+
+added early
--Purifiedextract
++-Microsomes
+++MessageRibosomestRNAs
Protein in supernatent
Protein in lumen of
microsomesProtein in supernatent
N
Protein in supernatent
From the previous experiment, Blobel demonstrated that the amino acidsequence at the beginning (N terminus) of exported proteins is recognized
by a complex.
This complex is required to get the protein into the lumen of ER.
To get into the lumen of the ER the protein has to be just beginning to be
translated.
Since not all exported proteins have the same N terminus, Blobelpredicted, like Millstein, whatever the sequence was, it would be later
cleaved.
Gunter BlobelNobel Laureate, 1999
+
Hydrophobic amino acids
N~20 amino acids
5’
“Signal Sequence”
Bacterium Eukaryotic Cell
NN
N
SRP
SignalSequence
Signal RecognitionParticle
SRP receptor“DockingProtein”
SRP receptor“DockingProtein”
Translocon
SRP receptor“DockingProtein”
SRP receptor“DockingProtein”
Signal peptidasecleaves off the signal
SRP receptor“DockingProtein”
Fully secreted Protein
SRP receptor“DockingProtein”
Signal peptidasecleaves off the signal
SRP receptor“DockingProtein”
Membrane protein
EUKARYOTIC CELL
Plasma membrane
EndoplasmicRecticulum
GolgiApparatus
CytoplasmOutside the Cell
TransportVesicles
EUKARYOTIC CELL
Plasma membrane
EndoplasmicRecticulum
GolgiApparatus
CytoplasmOutside the Cell
EUKARYOTIC CELL
Plasma membrane
EndoplasmicRecticulum
GolgiApparatus
CytoplasmOutside the Cell
EUKARYOTIC CELL
Plasma membrane
EndoplasmicRecticulum
GolgiApparatus
CytoplasmOutside the Cell
SecretoryVesicles
Plasma membrane
Endoplasmic Recticulum Golgi
Apparatus
Cytoplasm
Outside the Cell
SecretoryVesicles
Plasmamembrane
Endoplasmic Recticulum
Golgi Apparatus
Cytoplasm
Outside the Cell
Plasma membrane
EndoplasmicRecticulum
GolgiApparatus
Cytoplasm
Outside the Cell
Plasma membrane
EndoplasmicRecticulum
GolgiApparatus
Cytoplasm
Outside the Cell
Plasma membrane
Endoplasmic Recticulum
Golgi Apparatus
Cytoplasm
Outside the Cell
Plasma membrane
EndoplasmicRecticulum
Golgi Apparatus
CytoplasmOutside the Cell
Transportvesicles
SecretoryVesicles
cytoplasm
chromosome
Periplasm
Inner membrane
Outer membrane
β-galactosidase
lacZ
How were the Sec genes identified?
Bacterium
mRNA
Cytoplasm
chromosome
Inner membrane
β-galactosidase
lacZ
How were the Sec genes identified?
Active β-galactosidase is a tetramer.This cell can utilize lactose as a carbon source. LAC+
Gene encoding Exported Protein
lac Z gene5’ 3’
5’ 3’
5’ 3’
Gene Fusion
The 5’ end of the coding region of lac Z is fused to the 5’ end of a gene encoding
an exported protein including the signal sequence.
‘lac Z gene
5’ 3’
Gene Fusion
Where the 5’ end of the lac Z gene is fused to the 5’ end of a gene encoding an exported protein including the signal sequence.
This gene fusion results in a hybrid protein where theN-terminus of β-Galactosidase is fused with a signal sequence.
Signal Sequence
N C
β-galactosidase
Cytoplasm
chromosome
Periplasm
Inner membrane
Outer membrane
Hybrid protein
Gene fusion
The hybrid protein protein localizes to the membrane.
This cell is unable to utilize Lactose as a Carbon Source.
LAC-Cells with this gene fusion are…
Cytoplasm
chromosome
Periplasm
Inner membrane
Outer membrane
Gene fusion
LAC- LAC+
>95% of the Lac+ mutants have mutations …..linked to the gene fusion resulting in ….?
X Hybrid protein
X
X
X
X
Jon Beckwith
+
Hydrophobic amino acids
N
~20 amino acids
C
X
Cytoplasm
chromosome
Periplasm
Inner membrane
Outer membrane
Gene fusion
Hybrid protein
LAC- LAC+
X
X
XX
Cytoplasm
chromosome
Periplasm
Inner membrane
Outer membrane
Hybrid protein
Gene fusion
LAC- LAC+
X
X
XX
Conditional LethalHow to get get Mutations in essential genes
Temperature- sensitive
Cold-sensitive
20°C 37°C
Active Inactive
Active
Conditional LethalHow to get get Mutations in essential genes
Temperature- sensitive
Cold-sensitive
20°C 37°C
Active Inactive
Inactive Active
Sec ASec BSec DSec ESec GSec Y
Post-Translational
Post-translational
Cotranslational
Translationalstate ofprotein inchannel
What is theEnergy Source?
How does theprotein cross
the membrane?
Signal
Destination
GTP hydrolysisATP hydrolysisPowered bytranslation
Importinsdeliver to
Nuclear PoreComplex (NPC)
Chaperones bindProtein entersMito. Channel
SRP binds SSSRP binds DPProtein enters
channel
NuclearLocalizationSignal (NLS)7aa + charged
N-terminalAmphipathic
Helix20-50 aa
Cotranslational
Powered bytranslation
SRP binds SSSRP binds DPProtein enters
channel
Signal SequenceSignal Sequence
NucleusMitochondrionOutside the
cellPlasma
membrane
NOYesYesYesSignal Cleaved?
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