Lecture 3

• The ER organelle is fairly stable as a whole, but there is considerable localized movement of ER membranes
• The ER is an extensive lace-like network roughly divided into smooth and rough ER
• ER membrane proteins, “ reticulons ”, are responsible for membrane curvature
• ER targeting and translocation: Secretory Proteins

  1. Getting a protein into the ER lumen

  2. Need a targeting signal which is detected and sent to ER

  3. It is possible to artificially target non-ER proteins to the ER

  4. For secreted proteins, ER signal located at N-terminus of nascent polypeptide

  5. ER targeting needs to happen at the same time as protein synthesis – “Cotranslational Translocation”
• 1. way branching of ER tubules comes about from fusion of an extending tube with the side of another tubule
• Nascent proteins are folded, modified, and assembled within the ER lumen
• The ER plays important roles in protein quality control
• The Endoplasmic Reticulum (ER) is the largest continuous membrane structure in a cell
• The surface of Rough ER membranes is decorated with ribosomes and sites of protein synthesis (translation)
• ER membrane movement, extension, and retraction are coordinated with microtubules
• Dimerization of Atlastin-GTP link opposite membranes for ER membrane fusion
• Reticulons are inserted into ER membranes in a wedge-like conformation to curve the phospholipid bilayer
• Rough ER has a sheet-like structure or “cisternae” while Smooth ER has a highly branched, “tubular” morphology
• The life of secreted and plasma membrane proteins start at ER in the secretory pathway
• The ER is visible with staining with ER markers or electron microscopy but not easily visible under brightfield microscopy
• Phospholipid bilayers tend to be flat as curving the membrane requires energy expenditure
• Several reticulons give the sharp curvature of tubes and at the edges of ER sheets
• A small GTPase, Atlastin, is responsible for the 3-way branched structure of ER tubules
• Cleaved following targeting, i.e., not present on mature protein
• Insertion of Type II membrane proteins


  Type II membrane proteins do not have a cleavable N-terminal signal sequence, translation initially occurs in the cytoplasm, an internal ER targeting sequence is then recognized by SRP and directed to ER translocon, this internal targeting signal also doubles as an anchor signal: a "signal-anchor sequence", once the SA sequence is embedded, it is moved laterally along the bilayer and ribosome continues co-translation into ER lumen, reverse topology compared to Type I membrane proteins
• Cotranslational Translocation


  Signal peptidase in the ER cleaves the signal sequence off the polypeptide, polypeptide folds within the lumen of the RER
• ER membrane proteins are classified by topology (i.e., which way they face on the membrane)
• Cotranslational Translocation

  There is an adaptor complex, the signal recognition particle SRP, which binds to both the large ribosomal subunit and the signal sequence of the growing peptide
• ER targeting


  Needs to happen at the same time as protein synthesis - "Cotranslational Translocation"
• Insertion of Type III membrane proteins


  Same topology as Type I but translocation mechanism is similar to Type II, does not have a cleavable N-term signal sequence, uses a signal-anchor sequence but positioned very close to N-terminus, recognized by SRP, brought to translocon and anchored into the membrane but in reverse orientation to Type II (hence reverse topology), orientation of signal anchor sequence determined by position of positively charged flanking residues which prefer to remain on the cytosolic face, possible to artificially "flip" Type II and Type III membrane proteins by changing position of positively charged residues to reverse topologies
• ER-derived microsomes need to be present during protein synthesis to achieve membrane translocation
• Insertion of Type I membrane proteins

  Initial steps are identical to translocation of secreted proteins (i.e., SRP recognition and signal cleavage), insertion into the membrane requires a "stop-transfer anchor" (STA) signal, hydrophobic stretch of amino acids (20-25 aa) that embeds into the lipid bilayer
• Cotranslational Translocation

  There is a receptor for SRP in the ER membrane, translation is halted until the ribosome gets to ER translocon, docking of the SRP to its receptor opens up a channel allowing the translocation of the newly synthesized peptide
• Oligosaccharide binding membrane protein


  • Prevent misfolding
• Glycosylation


  Transfer of a chain of sugars (glycans) from a precursor catalyzed by glycosyltransferases
• Tail-anchored proteins use multiple stop-transfer anchor and signal-anchor sequences and a combination of Type I, II, and III mechanisms
• Type II and Type III protein topologies

  • Type II: Positive residues after the signal-anchor sequence
  • Type III: Positive residues before the signal-anchor sequence
• Disease associated with ER architecture: Hereditary Spastic Paraplegia
• Newly synthesized proteins in ER undergo several modifications

  Fold and assemble properly into mature complex prior to leaving ER (QC) to the golgi (logistics)
• ER membrane shape and complexity are required to maintain axon function
• Tail-anchored proteins do not use SRP directed co-translation
• Hereditary Spastic Paraplegia is a genetic condition caused by heritable autosomal mutations
• Defects in ER membrane shape cause retraction/loss of axons (axonopathy)
• Positively charged flanking residues


  • Prefer to remain on the cytosolic face
• Disulfide isomerase


  • Promote S-S bond formation