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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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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
  • Organelles are not made de-novo…expand existing structures

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


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

Bacterial Homologs:

With GTP molecules bound FtsY and Ffh recognize each other. Cleavage of GRPs leads to complex disassembly


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


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)


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

Translocation of type I membrane proteins require (1) signal sequence (cleaved) and (2) stop-transfer anchor sequence


Type II

Type III

Both of these protein types require just one internal hydrophobic signal-anchor sequence


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.


Why anchors are positive (+++)?

Membrane interior always has positive potential inside,

it would repel +++ clusters most effectively


+400 to +700 mV

Electric potential

dipoles created by lipid carbonyls and oriented water


GPI anchored proteins

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


Oxidized disulfide

Reduced dithiol form

PDI = protein disulfide isomerase


Proper folding of secreted protein

  • formation of correct S-S bonds
  • facilitate slow steps… peptidilyl-prolyl-isomerase (cyclophilin)
  • …cyclosporin A …tissue rejection