Lecture 20 Protein Targeting. The Rough ER: translocation and secretion reading: Chapter 13. Targeting of proteins: Location, location, location Constant growth required to maintain unique structure ...part of dissipative structure
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The Rough ER: translocation and secretion
reading: Chapter 13
A 16-30 residue hydrophobic signal sequence directs the ribosome to the ER
The hydrophobic core of the signal sequence contains one or more “+” charged residue
With GTP molecules bound FtsY and Ffh recognize each other. Cleavage of GRPs leads to complex disassembly
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
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?
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)
Translocation of type I membrane proteins require (1) signal sequence (cleaved) and (2) stop-transfer anchor sequence
Both of these protein types require just one internal hydrophobic signal-anchor sequence
Type I…targeting sequence and stop-transfer sequence
Type II, III…internal sequences…oriented by positive cluster…why?
Type IV…multipass…target…stop transfer…etc.
Membrane interior always has positive potential inside,
it would repel +++ clusters most effectively
+400 to +700 mV
dipoles created by lipid carbonyls and oriented water
Purpose? …Lateral diffusion….?
Transfer of type 1 protein
N-linked poly-sugar chains are synthesized on Dolichol phosphate utilized as an attachment anchor
Dolicol = 75-90 carbon isoprenoid lipid
Oligosacharides attached to proteins help folding through specific associations with lectins
Removal and re-attachment of one glucose residue acts as a ‘quality control’ step in the process of folding (see text).
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
Reduced dithiol form
PDI = protein disulfide isomerase
DPI = protein disulfide isomerase