Cytoplasm chromosome Periplasm Inner membrane Outer membrane Outside the cell BACTERIAL CELL mRNA protein MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel
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Cytoplasmchromosome
PeriplasmInner membrane
Outer membrane
Outside the cell
BACTERIAL CELL
mRNA
protein
MIT Biology Department
7.012: Introductory Biology - Fall 2004
Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel
EUKARYOTIC CELL
Mitochondrion
Plasma membrane
Nucleus
Endoplasmic
Recticulum Golgi
Apparatus
Cytoplasm
Outside the Cell
N
Eukaryotic
Cell
Bacteria
Insulin
Growth Factors
Antibodies
Insulin Receptor
Growth Factor
Receptors
Histidine synthesis
Lactase
Glycolysis Enzymes
Cyclins
ToxinLactose Receptor��galactosidase
Fully
SecretedProtein
(Outside the Cell)
Membrane
Protein
Cytoplasmic
Protein
Examples
George Palade
Images removed due to copyright reasons.
Images removed due to copyright reasons.
Hamster pancreatic Nucleus
Mitochondrion
Earliest Time point
Next
observed
location
Location
After
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 of
light chains. Further experiments led us to propose
the extra N-terminal sequence was a signal for
vectorial transport across the membrane during
protein synthesis. That was the first evidence which
indicated that the signal for secretion was an N-terminal
segment, rapidly cleaved during protein synthesis.”
FROM NOBEL LECTURE 1984
Image removed due to copyright reasons.
CytoplasmicExtracts
N
Messenger RNARibosomes &
charged tRNAs
Microsomes(RER vesicles)
in vitro
Blobel
Image removed due to copyright reasons.
+added late
+
+
+added early
--Purified
extract
++-Microsomes
+++Message
Ribosomes
tRNAs
Protein in
supernatent
Protein in
lumen of
microsomes
Protein in
supernatent
N
Protein in
supernatent
From the previous experiment, Blobel demonstrated that the amino acid
sequence 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, Blobel
predicted, like Millstein, whatever the sequence was, it would be later
cleaved.
Gunter Blobel
Nobel Laureate, 1999
Image removed due to copyright reasons.
+
Hydrophobic amino acids
N
~20 amino acids
5’
“Signal Sequence”
Bacterium Eukaryotic Cell
N
N
N
SRP
Signal
SequenceSignal Recognition
Particle
SRP receptor
“Docking
Protein”
SRP receptor
“Docking
Protein”
Translocon
SRP receptor
“Docking
Protein”
SRP receptor
“Docking
Protein”
Signal peptidase
cleaves off the signal
SRP receptor
“Docking
Protein”
Fully secreted Protein
SRP receptor
“Docking
Protein”
Signal peptidase
cleaves off the signal
SRP receptor
“Docking
Protein”
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
Secretory
Vesicles
Plasma membrane
EndoplasmicRecticulum Golgi
Apparatus
Cytoplasm
Outside the Cell
Secretory
Vesicles
Plasmamembrane
EndoplasmicRecticulum
GolgiApparatus
Cytoplasm
Outside the Cell
Plasma membrane
EndoplasmicRecticulum
GolgiApparatus
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
CytoplasmOutside the Cell
Transportvesicles
SecretoryVesicles
cytoplasm
chromosome
Periplasm
Inner membrane
Outer membrane
�-galactosidase
lacZ
How were the Sec genes identified?
Bacterium
mRNA
Cytoplasm
chromosome
Inner membrane
�-galactosidase
lacZ
How were the Sec genes identified?
Active �-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 �-Galactosidase is fused with a signal sequence.
Signal Sequence
N C
�-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