L9 secretory pathway proteins(MP3)

• In the secretory pathway, the components are continuous and different from those in the cytosol
• Phospholipids are synthesized in the cytosolic layer of the ER, extending from other membranes in the cell
• Proteins in the secretory pathway are encoded by genes in the nucleus, transcribed into mRNA in the cytosol, and then produce a protein with signals determining their destination
• The amino acid sequence of a protein determines its folding, post-translational modifications, and destination through specific signals
• During protein synthesis, as the peptide is being synthesized, it moves to the ER, explaining why many ribosomes are attached to the ER
• All proteins of the secretory pathway are inserted into or across the ER before moving to the golgi, plasma membrane, or lysosome
• The outer and inner nuclear membranes are continuous with the ER
• Targeting signals or signal peptides are sequences within the nascent peptide that are independent from the area of the protein with the biochemical function
• Hypothesis for secretory proteins and signaling:

  • Observed that secretory proteins are larger before maturing, indicating an extra sequence in the protein that acts as a signal
  • Experiment involved creating a tube with cytosol and ribosomes, synthesizing RNA encoding a secretory pathway protein, and observing the role of the signal peptide in protein synthesis.
  • found that the translocon (a pore in the ER that recognizes the signal) allows the protein to be synthesized in the ER. a peptidase removes the signal.
• Ribosome exit tunnel:

  • Contains small and large subunits with a tunnel in the large subunit where the nascent polypeptide accumulates before leaving the ribosome
  • Interaction between the signal peptide and the translocon occurs as the peptide exits the tunnel
• Targeting steps and components (SRP, SRP-R, and translocon):

  
  

  1. Recognition of the signal in the protein involves the ribosome, signal recognition particle (SRP), and SRP receptor

  2. Connection of the protein to the ER includes the SRP receptor linking the ribosome to the translocon

  3. Translocation of the protein into or across the membrane requires the energy of the translating ribosome
• Components of target signal:

  • Proteins enter or cross the ER cotranslationally
  • Signal peptides usually have 8-16 hydrophobic residues surrounded by polar amino acids, often at the N-terminus
  • Signal anchors are signal peptides that become transmembrane helices integrated into the transmembrane
• SRP:

  • A ribonucleoprotein with 6 protein subunits and 1 RNA subunit
  • Recognizes ribosomes and slows down translation when it identifies the hydrophobic characteristic of the ER signal
  • it has the region that recognizes ribosome (which stimulates GTPase activity of the receptor) and region that is the translation regulatory domain (that will slow down translation)
  • SRP will sample all the nascent polypeptides, and when it has the hydrophobic characteristic of the ER signal, then pocket of SRP will bind to them. this leads to a conformational change, binding itself to the ribosome
• SRP-R GTPase activity:

  • when the SRP-R is bound to SRP, it is also bound to ribosome and GTP.
  • SRP-R hydrolyzes GTP when bound to SRP, leading to the disassociation of SRP and SRP-R
• ER translocon:

  • The yeast and mammalian translocon are similar, with the yeast known as sec61
  • The sec61 complex has two parts forming both sides of the aqueous pore, allowing the nascent peptide to pass through when the signal peptide pushes the plaque aside
• some of the signals for the secretory pathway can become a transmembrane protein via synthesis of a hydrophobic patch 18-24 amino acids long that is a signal to stop inserting the protein into the ER, causing it to anchor into the membrane during translation instead.
• the types of transmembrane proteins that come from signals of the secretory pathway are (1) type 1, with N-terminus in the ER and c-terminus in the cytosol. (2) type 2, with c-terminus in the lumen and n-terminus in the cytosol. (3) multi-pass TM proteins, with many different patches integrated into the membrane.
• positive charges will always be in the cytosol and negative charges will be in the lumen
• to ensure the quality of secretory proteins in the ER, they can under go n-linked glycosylation (a PTM), degradation, or be degraded by ER chaperones
• the quality control mechanism of proteins made in the ER of n-linked glycosylation

  • motif for glycosylation is: asn-a-ser/thr
  • all proteins in the secretory pathway get this glycan added (has 2 acetylglucosamines, different varieties of mannose, 3 glucose molecules) which ensures that these proteins are only for the secretory pathway
  • oligosaccharyl transferase is the enzyme that adds this sugar (called OST), a transmembrane protein
  • most asparagines in this motif will receive this glycan.
• the purpose of the addition of a glycan in n-linked glycosylation is because the addition of the sugar (1) stabilizes the native state, (2) protect against proteases (3) and have a PTM that will change the signaling properties.
• secretory proteins in the ER that need to be degraded will seep out into the cytosol; another degradation system is digestion by proteases inside lysosome
• disulfide bonding among the cysteins is the other PTM that is done in the ER, it is a covalent modification