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?