Lecture 12 Protein Sorting II 2024 PDF

Protein Sorting: Endoplasmic Reticulum RER in pancreatic acinar cell 1  Movement of Proteins Between Compartments 1. Gated transport: nucleus 2. Transmembrane transport: mitochondria and ER 3. Vesicular transport: secretory pathway...

Protein Sorting: Endoplasmic Reticulum RER in pancreatic acinar cell 1  Movement of Proteins Between Compartments 1. Gated transport: nucleus 2. Transmembrane transport: mitochondria and ER 3. Vesicular transport: secretory pathway 2  Protein Sorting to the ER ER is a network of branching tubules. A membrane separates ER lumen from cytosol and mediates transfer of proteins. Major site of protein and lipid biosynthesis. Transmembrane vs. water-soluble proteins. Cotranslational translocation (primarily). Many of the cell’s proteins must first pass through the ER. 3 Figure 12-35 Molecular Biology of the Cell (© Garland Science 2008)  Role of the Ribosome Ribosomes are protein complexes that associate with mRNA and catalyze synthesis of protein. Ribosomes are either free (cytosolic) or membrane- bound (i.e. rough ER). Ribosome with polypeptide chain will be directed to ER membrane only if the appropriate signal sequence is present (otherwise it will remain in cytosol). 4  Signal Sequence As described for mitochondrial translocation, a signal sequence guides polypeptide chain to ER. Cleaved by signal peptidase. However, it’s a little more complicated than that. SRP and receptor. 5 SRP = signal recognition particle  Signal Recognition Particle SRP cycles between ER membrane and cytosol. Reversibly binds to ribosome and SRP receptor. SRP binding site is a methionine- rich “pocket” that accommodates many signal sequence types. SRP receptor is in ER membrane. 6 Ribosomes are Directed to the ER Membrane 1 3 2 4 7 Components: ribosome, tRNA, mRNA, growing polypeptide with SS Sec61 Protein Translocator Sec61 complex forms an aqueous pore in ER membrane. Aligns with nascent polypeptide. Signal sequence triggers opening of pore after its release from SRP. 8  Translocation of Soluble Proteins Signal sequence is a “start-transfer signal”. Signal sequence is cleaved by a peptidase at a specific cleavage site. Release of polypeptide (to lumen) and SS (to membrane). Note: ribosome, SRP and SRP receptor not shown in these models for clarity. Fig. 12-46 Alberts 9  Single-Pass Transmembrane Proteins (3 Models) A “stop-transfer signal” will bind to the translocon and prevent further translocation. Released into the membrane as an  helix. Model 1 10  Internal signal (start) sequence is recognized and initiates translocation.  helix not cleaved but will remain in membrane. **Final orientation of the protein is determined by the polarity of the SS. (-) end of SS always enters the translocon first. Model 2 11  Entry of (-) end of SS causes reorientation of the growing polypeptide chain Model 3 12  Double-Pass Membrane Proteins Internal signal sequence initiates translocation. Stop-transfer signal stops translocation. Both sequences are  helices and are released to the membrane. 13  Multipass Membrane Insertion Insertion of further hydrophobic protein segments is intermittently reinitiated by additional start and stop transfer signals. Segments bind to the same translocon and are released as  helices to the membrane. e.g. insertion of rhodopsin into the ER membrane. Fig. 12-50 Alberts 14  Posttranslational Translocation Not all proteins are translocated to the ER lumen by a cotranslational mechanism. Those formed by free ribosomes are translocated by a posttranslational mechanism, e.g. SNARE, Bcl2 family. In eukaryotes, accessory proteins (receptors) associate with Sec61 and span the ER membrane. Molecular chaperones (BiP) bind to polypeptides and pull chain through, as in mitochondrial ratchet model. Note: the signal sequence is present but not illustrated in this model. 15 Fig. 12-45B Alberts  Translocated Polypeptides Translocated polypeptide chains are folded in the ER. These proteins may remain, or continue to other destinations. 3 basic steps: – 1) Glycosylation – 2) Folding – 3) Export; or if not folded properly…degradation of misfolded proteins 16  Protein Glycosylation Oligosaccharide transferred from the lipid, dolichol, to asparagine residue of polypeptide. Reaction catalyzed by oligosaccharyl transferase. Protein is then marked for folding. 17 Role of Glycosylation in Protein Folding Calnexin binds to incompletely folded proteins in the ER. Glucosidase “trims”, and glucosyl transferase adds glucose. Cycle continues until protein folded properly. (glucosidase) 18  Misfolded Proteins Misfolded proteins are “dislocated” through Sec61. Chaperone, lectin, disulfide isomerase deliver to translocator. Translocator E3 ligase mediates ubiquitination. ATP hydrolysis pulls chain. Deglycosylation by N-glycanase. Degradation. 19  Things to Consider... 1. Why are molecular chaperones usually not needed for delivery of polypeptide chains to the ER? 2. What is the difference between posttranslational and cotranslational mechanisms of translocation? 20