Protein sorting in a eukaryotic celldalbey/courses/Chem763... · Protein Translocation Across the...
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Protein sorting in a eukaryotic cell
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Questions in Protein Insertion into the ER
• How are proteins targeted correctly to the ER?• How do hydrophilic regions of secreted proteins
cross the apolar lipid bilayer?• How are hydrophilic domains of membrane
proteins translocated across the membranes?
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George Palade
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Intracellular Protein Traffic
Günter Blobel, M.D., Ph.D. Investigator — Cell Biology
Dr. Blobel is also John D. Rockefeller, Jr., Professor of Cell
Biology at the Rockefeller University. He received his M.D.
degree from the University of Tübingen and his Ph.D. degree in
oncology from the McArdle Laboratory at the University of Wisconsin–Madison, where he
worked with Van Potter. Thereafter, he did postdoctoral work with
George Palade at Rockefeller. Dr. Blobel is a member of the National Academy of Sciences and of several other distinguished societies. He has received many honors,
including the 1993 Lasker Award in basic medical research, the 1996 King Faisal International Prize for
Science, and the 1999 Nobel Prize in physiology or medicine.
HHMI Investigators
Research field Cell Biology Genetics Immunology Neurosciences Structural Biology Investigator Site/Related Links Science Publications Research In Progress Home News HHMI Home
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Nobelförsamlingen
Karolinska Institutet
Press Release October 11, 1999
The Nobel Assembly at the Karolinska Institute has today decided to award the 1999 Nobel Prize in Physiology or Medicine to
Günter Blobel
for the discovery that
"proteins have intrinsic signals that govern their transport and localization in the cell"
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Signal Sequence Hypothesis
•Signal peptidase (leader peptidase) cleaves N-term signal peptides
•N-term signal sequences are ~10-27 amino acids long•Unidirectional translocation is mediated by “signal sequences”
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The Substrate Showing the Signal Sequence
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Mammalian co-translational translocation(Corsi and Schekman, 1996, JBC, 271, 30299)
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The Ffh M-domain/4.5 S complex(Batey et al., Science 287:1232)
RNA
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Current Model of Protein Translocation(Co-translational)
•mRNA encoding the protein destined for the ER attached to a “free”ribosome in the cytosol
•The N-terminal signal sequence of the nascent precursor chain emerges from the large ribosomal subunits
•After most or all of the signal sequence has emerged, it binds a cytosolicribonucleoprotein complex termed “signal recognition particle” or SRP
•Interaction with the SRP stops further elongation of the polypeptide chain. GDP bound to SRP is exchanged for GTP at this step.
•The arrested complex binds to a specific 72 kd receptor (SRP receptor) which protrudes from the cytosolic face of the ER.
•After the signal sequence of the exported protein has emerged on the lumenal side of the ER, the signal peptide is cleaved off by signal peptidase.
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Translocation Across the ER MembraneCo-translational
•Energy source- GTP hydrolysis
•Proteins move across the ER membrane through an aqueous pore at sites called translocons.
•Translocation components (Sec61αβγ or SecYEG)
•Ribosome binding to the translocon prevents ion and small molecule movement through the aqueous pore.
•In co-translational translocation, the ribosome binds to the translocon to form a single aqueous conduit that stretches across the ER membrane and is sealed off from the cytoplasm
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Corsi, A. K. et al. J. Biol. Chem. 1996;271:30299-30302
Yeast post-translational translocation
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Protein Translocation Across the MembranePost-translational
•Energy Source is ATP hydrolysis.
•Cytosolic chaperones- Ssa1p (DnaK); Ydj1p (DnaJ)- Ssa1p is an ATPase.
•Post-translational targeting to the membrane- Require components Sec62p, Sec71p, and Sec72p.
•ER lumenal chaperone- Bip (DnaK)- an ATPase; the ER membrane component Sec63 has the DnaJ domain
•Sec translocon is Sec61αβγ
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ER-membrane
proteins involved in
protein translocation
(Rapoport et al, 1996, Annu. Rev.
Biochem., 65, 271)
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View of the protein conducting channel
Rapoport and coworkers
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Art Johnson (Texas A&M) Protein Transport Gordon conference 2005
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The ribosome-Sec61p complex(Beckmann et al., Science 278:2123)
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The translocation channel
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Tom Rapoport(Harvard Medical School)
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2D EM of E. coli SecY complex
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General Architecture of the SecY complex
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Structure of the channel: gating in two directions
TIBS 2004
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Distribution of Polar Residues
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The Channel Pore
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Sec61/SecY complexes in vivo
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Formation of a consolidated pore between two Sec channels?
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Different Sizes of the Translocation Component
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Major Points
• Structure of Sec61αβγ (SecY complex) from the archaeon Methanococcus janaschiiwas solved 3.2 Angstrom resolution
• Sec61α (SecY) has two symmetrical halves (Transmembrane segments 1-5 and 6-10)
• Only one copy of Sec61αβγ not multicopies• Aqueous channel with a constricted
hydrophobic pore• Pore is plugged by a helix
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A Ribosome at the End of the Tunnel
(Powers and Walter, 1997, Science, 278, 2072)