7 - endomembrane system ii

Question 1

what is the ER?

Answer 1

• ER is the starting point for the secretory and biosynthetic pathways
• • site of protein and lipid synthesis, protein folding, and processing
• highly complex network of membrane enclosed, rod like tubules and sheet like cisternae
• • largest surface area of any organelle
• lumen: aqueous space inside of ER tubules and cisternae
• tubules and cisternae shapes are mediated by reticulons
• • reticulons: unique ER integral membrane proteins that have a hair pin secondary structure and regulate ER membrane curvature and overall shape
• highly dynamic network
• • ER tubules and cisternae are always moving, dividing or fusing, and growing or shrinking
• has multiple subdomains

Question 2

what are ER subdomains?

Answer 2

• distinct regions of the ER network that have different morphologies and functions
• • e.g. rough ER and smooth ER
• • • RER: mostly cisternae bound by ribosomes, protein and membrane phospholipid synthesis
• • • SER: mostly curved tubules without ribosomes, stores Ca2+ and makes hormones
• more than 20 other ER subdomains
• • have unique complements of proteins and membrane lipids that control their specific functions
• other subdomains
• • nuclear envelope
• • • outer nuclear membrane is continuous with RER, has nups and attached ribosomes
• • mitochondria and plasma membrane associated membranes (MAM and PAM)
• • • regions of ER that touch the mitochondria or pm, respectively
• • • exchange membrane proteins and lipids
• • ER exit sites (ERES): regions where transport vesicles bud off the ER and go to the golgi

Question 3

what is the rough ER?

Answer 3

• one of two main sites for translation in the cell
  1. free ribosomes in the cytoplasm
• • fate of nascent properly folded soluble or membrane protein in the cytoplasm:
• • • remains in the cytoplasm
• • • • e.g. glycolytic enzyme
• • • or targets to the proper intracellular destination
      
      2. ER membrane bound ribosomes
• • fate of the nascent properly folded soluble or membrane protein in the RER:
• • • remains in the RER or goes to another ER subdomain
• • • localizes to other ER derived organelles
• • • or goes from the ER onto another part of the EMS via transport vesicles

Question 4

what are the steps of cotranslational translocation?

Answer 4

1. an mRNA is translated on a free ribosome in the cytoplasm
   - - N terminus of the growing polypeptide emerges from the ribosome
   - - it has a signal sequence of 8 to 15 hydrophobic amino acids that are the RER targeting signal
2. exposed signal sequence is recognized by the SRP
   - - SRP then binds to the ribosome and stops protein translation
3. SRP targets the complex to the surface of the ER and binds to the SRP receptor on it
   - - SRP receptor: heterodimeric ER integral membrane protein complex
   - - cytoplasmic facing domains serve as a docking site for the SRP
   - - interaction between SRP and its receptor is strengthened by both of them binding to GTP
4. GTP hydrolysis causes the SRP to be released from its receptor which can be used for another round of import
   - - at the same time, the nascent polypeptide and ribosome go to the cytoplasmic side of the Sec61 translocon
   - - - Sec61: multi protein complex that has several ER integral membrane protein subunits that make an hour lass shaped aqueous channel
5. N terminus of the polypeptide goes into the Sec61 translocon channel
   - - then translation resumes and the elongating polypeptide starts to enter the ER lumen through the translocon
   - - - since the passage of the polypeptide through the translocation is driven by its translation, that’s why it’s called cotranslational translocation
6. as the N terminal signal sequence enters the ER lumen, it is cleaved by the signal peptidase and degraded
   - - signal peptidase: ER integral membrane protein/protease associated with the translocon
   - - - the catalytic domain of the peptidase faces the ER lumen
   - - - it recognizes the cleavage sequence at the C terminal end of the signal sequence
7. then cotranslational translocation of the polypeptide continues into the ER lumen
8. once translation is done, ribosome releases from the translocon
   - - the rest of the protein goes into the ER lumen
   - - the pore plug moves back into the channel to close the translocon

Question 5

what is the sec61 translocon?

Answer 5

• hour glass shaped tranlocon channel has a pore ring
• • pore ring has 6 hydrophobic amino acids at the narrowest diameter of the channel
• • act as a gate that seals the channel to ions and small molecules
• • translocon channel is also blocked by a short alpha helix plug
• • it’s the second gate keeping mechanism as it maintains organelle compartmentalization
    during protein translocation, the growing polypeptide forces plug away from the channel

Question 6

what is cotranslational insertion?

Answer 6

• cotranslational insertion if an integral membrane protein into the RER
• most membrane proteins are also made on membrane bound ribosomes at the RER
• • including resident membrane proteins of the ER and all other pst ER compartments of the EMS
• • • golgi, lysosomes, pm, etc.
• ER membrane protein insertion is similar initially to soluble protein import into ER lumen
• • except there are important mechanistic differences resulting in the mature membrane protein being integrated in the ER membrane with proper topology
• • topology: the way it is arranged

Question 7

what are the integral membrane topologies?

Answer 7

• membrane protein topology: # of membrane spanning domains and orientation
• • transmembrane domain (TMD): typically an alpha helical 16 to 25 hydrophobic amino acid chain
• • • energetically favourable within hydrophobic interior of phospholipid bilayer
• several different classes of integral membrane proteins are synthesized at the ER
  
  • type i 1 TMD N in, C out signal sequence
    type ii 1 TMD C in, N out no signal sequence
    type iii 1 TMD N in, C out no signal sequence

Question 8

what is the type i membrane protein?

Answer 8

• N in ER lumen, C in cytosol
• nascent polypeptide/ribosome complex goes to and associates with translocon
• N terminus enters ER lumen and is cleaved by signal peptidase
• cotranslational translocation continues
• eventually, first or only hydrophobic TMD enters the translocon
• • TMD serves as an internal stop transfer anchor (STA) sequence
• • stops further translocation of polypeptide through the translocon
• STA sequence moves laterally out of translocon and becomes anchored in adjacent phospholipid bilayer
• as translation continues, elongating polypeptide extends into the cytosol
• when translation is done, ribosome is released and protein diffuses away laterally in the membrane bilayer

Question 9

what is the type ii membrane protein?

Answer 9

• N in cytosol, C in ER lumen
• opposite orientation to type i
• no N terminal signal sequence
• • has an internal signal anchor (SA) sequence
• first and only TMD works both as a signal sequence for binding SRP and mediating nascent polypeptide/ribosome complex to bind to the translocon and as a membrane anchor
• protein’s internal SA sequence enters translocon
• • then it is flipped so N terminus of the polypeptide faces the cytosol
• • orientation is mediated by positive charged amino acid residues upstream of SA sequence
• • positively charged AAs next to SA sequence determine orientation of most membrane proteins made at the ER
• • “positive outside rule”
• as translation continues, elongating polypeptide goes into the ER lumen
• the SA sequence moves laterally out of the translocon and becomes anchored in the adjacent membrane bilayer
• when translation is complete, the ribosome is released and the entire protein diffuses away laterally in the ER membrane bilayer

Question 10

what is the type iii membrane protein?

Answer 10

• N in ER lumen, C in cytosol
• same orientation as type i but has an internal SA sequence
• SRP dependent targeting to and insertion into translocon is similar to type ii membrane protein but the positively charged AAs are located downstream of the SA sequence
• as translation continues, elongating polypeptide extends into the cytoplasm
• when translation is done, the ribosome is released and the protein diffuses away laterally in the membrane bilayer