Major Protein-sorting pathways in eukaryotic cells

Key elements for the Protein targeting events

1. Signal sequence: the amino acid sequence of

a protein that provides the information to
target the protein to a particular organelle.

2. Receptors for the signal sequences.

3. Translocation channels that allow transfer of

proteins across the membrane bilayer.

4. Energy that drives unidirectional transfer

across the membrane.
Where are the endoplasmic reticulum, Golgi, and
mitochondria?
Synthesis of secretory proteins and their cotranslational
translocation across the ER membrane

Wenxia:
Wenxia:
1.1. ER
ERsignal
signalsequence
sequence
emerges
emerges
2.2. The
Thebinding
bindingby
byaa
signal-recongition
signal-recongition
particles
particles(SRP)
(SRP)
3.3. SRP
SRPdelivers
deliversthe
the
ribosome/nascent
ribosome/nascent
polypeptide
polypeptide
complex
complextotothe
theSRP
SRP
receptor
receptorininthe
theERER
membrane,
membrane,and and
GTP binding
GTP binding
Signal-
recognition
particles

Wenxia:
Wenxia:
1.1. Six
Sixsubnits
subnits
2.2. All
Allproteins
proteinsbinding
bindingtoto
300-nucleotide
300-nucleotideRNA
RNA
except p54
except p54
3.3. P54
P54bind
bindtotoER
ERsignal
signal
sequences and have
sequences and have
hydrophobic
hydrophobicbinding
binding
groove
groove
 Translocon

Wenxia:
Wenxia:
1.1. First
Firstfound
foundininyeast
yeastby
by
mutations
mutations
2.2. Sec61
Sec61complex,
complex,
consisting
consistingofof33proteins
proteins
3.3. Sec61a
Sec61acontacts
contacts
translocating
translocating
polypeptide
polypeptidechain
chain
4.4. AAcylinder
cylinder5-6
5-6nm
nmhigh,
high,
8.5
8.5 nm in diameter,and
nm in diameter, and
pore 2 nm diameter
pore 2 nm diameter
5.5. Open
Openandandclose
closeofof
translocon
transloconisisregulated
regulated
Major topological classes of integral membrane
proteins synthesized on the rough ER
Wenxia:
Wenxia:
Hydrophobic
Hydrophobic
Synthesis and insertion into the ER membrane
transmembrane
transmembrane
segment
segmentisisthe
the
stop-transfer
stop-transfer
of type I single pass proteins
anchor
anchorsequence
sequence
 Type II single-
pass proteins

Experiments:
Experiments:
Mutating
Mutatingthe the
positive charged
positive charged
amino
aminoacids
acids
change
changethe the
orientation
orientationofofthe
the
protein.
protein.
Arrangement
of topogenic
sequences of
different
membrane
proteins
GPI anchored proteins
Mitochondrial
 Protein import into
the mitochondrial
matrix
Tom-translocon of outer
membrane
Tim-translocon of the inner
membrane

Wenxia:
Wenxia:
Targeting
Targetingsignals
signals
Signal
Signalreceptor
receptor
Translocation
Translocation
channel
channel
Energy
Energy
Transporting protein from the cytosol to the
inner mitrochondrial membrane
Transporting proteins from the cytosol to the
mitochondrial intermembrane space
How to analyze protein movement into
membranes and organelles

The in vitro system

 How to identify signal sequences

Genetic manipulations
1. Sequence homology
2. Targeted mutagenesis
3. Fusion proteins
Hydropathy profiles
How to identify a translocon component that
contacts nascent secretory proteins

Chemical
cross-linking