Download - Lecture 20 Protein Targeting The Rough ER: translocation and secretion reading: Chapter 13
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Lecture 20
Protein Targeting
The Rough ER: translocation and secretion
reading: Chapter 13
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Targeting of proteins:
•Location, location, location
•Constant growth required to maintain unique structure
•...part of dissipative structure
•Organelles are not made de-novo…expand existing structures
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Secreted proteins are sequestered from the cytoplasm in microsomes
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A 16-30 residue hydrophobic signal sequence directs the ribosome to the ERThe hydrophobic core of the signal sequence contains one or more “+” charged residue
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Rough ER
•Targeting sequence on protein…not mRNA
•Must be translated to be read
•Translation must stop…wait to dock with site on RER (why?)
•Signal-recognition particle, SRP, stops translation and provides for delivery …mailman…recognizes 3 structures
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Bacterial Homologs:With GTP molecules bound FtsY and Ffh recognize each other. Cleavage of GRPs leads to complex disassembly
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Sec61 (SecY) from Methannococcus jannaschii
A ring of Isoleucine residues forms a hydrophobic ‘seal’ in the middle of the channel
The ‘plug’ helix moves out during translocation
Lateral exit into the bilayer can be permitted if blue helices separate
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Liposome reconstitution experiments have demonstrated that Sec61, SPR receptor, and the nascent protein complexed with ribosome and SPR are absolutely required for translocation.
No additional energy is required for translocation
Can proteins be translocated not co-translationally, but post-translatonally?
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Yes, in yeast successive binding of BiP-ADP makes transport unidirectional
BiP is a chaperone protein
The signal sequence is cleaved soon after (in both mechanisms)
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Translocon is dynamic
•Soluble proteins end up in “exoplasmic space” …topologically equivalent to the extracellular space
•Transmembrane segments of membrane proteins move sideways into RER membrane
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Types of transmembrane proteins
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Translocation of type I membrane proteins require (1) signal sequence (cleaved) and (2) stop-transfer anchor sequence
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Type II Type III
Both of these protein types require just one internal hydrophobic signal-anchor sequence
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Threading membrane proteins through the membrane
Type I…targeting sequence and stop-transfer sequence
….SRP/receptor…cleaved sequence
Type II, III…internal sequences…oriented by positive cluster…why?
Type IV…multipass…target…stop transfer…etc.
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Ele
ctric
pot
entia
lmembrane
dipoles created by lipid carbonyls and oriented water
Membrane interior always has positive potential inside,it would repel +++ clusters most effectively
Why anchors are positive (+++)?
+400 to +700 mV
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Glucosylphosphatidyl inositol (GPI) anchor
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GPI anchored proteins
Purpose? …Lateral diffusion….?
…Targeting?
Transfer of type 1 protein
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GLUT1 protein
Hydrophobicity profiles of primary sequences
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N-linked poly-sugar chains are synthesized on Dolichol phosphate utilized as an attachment anchor
Dolicol = 75-90 carbon isoprenoid lipid
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Oligosacharides attached to proteins help folding through specific associations with lectinsRemoval and re-attachment of one glucose residue acts as a ‘quality control’ step in the process of folding (see text).
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Proper folding of Hemagglutiin (HAo) occurs in the presence of chaperones (Bip) and two types of lectins (Calnexin and Calreticulin). The folded structure works as a pre-loaded spring in the mechanism of Influenza virus membrane fusion mediated by HAo
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Reduced dithiol formOxidized disulfide
PDI = protein disulfide isomerase
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DPI = protein disulfide isomerase
What if the protein has more than one disulfide bond?
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Proper folding of secreted protein
•formation of correct S-S bonds
•facilitate slow steps… peptidilyl-prolyl-isomerase (cyclophilin)
…cyclosporin A …tissue rejection