2/22/15 1 Protein sorting Reading: ECB3 500-509 or ECB4 492-502 Learning Objectives: • Understand the mechanisms by which proteins are targeted to different cellular compartments • Understand the concept of signal sequences • Understand how the Ran-GTPase cycle leads to nuclear import of proteins • Understand how proteins are targeted to mitochondria and chloroplasts post-translationally • Understand the mechanism by which SRP couples translation and translocation of proteins into the endoplasmic reticulum • Understand how proteins that have many transmembrane regions are inserted into the endoplasmic reticulum Prelim 1, Wednesday, 9:05-9:55 We will use SCANTRON “bubble” sheets Bring a couple of #2 pencils and a good eraser. We will not return SCANTRON sheets, so mark answers on your exam if you want to keep track Office Hours Monday Feb. 23 Joel 1:00-2:00 107 Stimson Kevin M. 2:00-3:00 107 Stimson Tuesday Feb. 24 Kevin H. 1:00-2:00 107 Stimson Dante 2:00-4:00 107 Stimson2/22/15 2 IMPORTANT – EXAM LOCATIONS If your last name starts with A, B, C or D, you will be taking the exam in 228 Malott Hall If your last name starts with E-Z, you will be taking the exam in Call Auditorium You MUST go to the correct location! Tower Road Last Lecture……………2/22/15 3 How do proteins know where to go? The sorting problem Figure 12-18 15-2 Essential Cell Biology Figure 15-5 Essential Cell Biology Organelles import proteins by several mechanisms cytosol - 2 types of glucose transporters enable gut epithelial cells to transfer glucose across the epithelial lining of the gut. - Na+ is pumped out by Na+ pumps in the basal and lateral plasma membrane. - how animal control the pH in their cytosol with use of Na+ to pump H+ out of the cell- all processes require energy - protein remains folded during transport in mechanisms 1 and 3 but has to be unfolded in 21: - proteins moving from the cytosol into nucleus - pores function as selective gates that actively transport specific macromolecules - pores also allow free diffusion of smaller molecules2: - proteins moving from cytosol to ER, mitochondria, or chloroplasts transported by protein translocators - protein needs to unfold for it to move across the membrane through translocators 3: - proteins moving onward from the ER - transport vesicles: pinch off from the membrane of 1 compartment and then fuse with the membrane of a 2nd compartment - vesicles deliver soluble cargo proteins2/22/15 4 Figure 15-6 Essential Cell Biology Signal sequences are necessary and sufficient to direct a protein to its destination A signal sequence is both NECESSARY and SUFFICIENT to target a protein to the correct organelle. Table 15-3 Essential Cell Biology Signal sequences direct proteins to the correct compartment ‘Molecular ZIP codes’ A: - proteins destined for ER have a N-terminal signal sequence - proteins destined to stay in the cytosol lack any signal sequenceB: - if signal sequence is removed from an ER protein and attached to a cytosolic protein, both proteins are relocatedER signal sequence removed from ER protein and attached to cytosolic protein2/22/15 5 Today’s topics 1. Transport into the nucleus 2. Transport into mitochondria 3. Transport into the endoplasmic reticulum Figure 15-5 Essential Cell Biology Nuclear Transport cytosol2/22/15 6 Figure 15-7 Essential Cell Biology Architecture of the nuclear envelope Figure 15-8b Essential Cell Biology Electron micrographs of nuclear pores face-on view side view 75 nm - outer nuclear membrane is continuous with the ER membraneNuclear envelope: - encloses the nuclear DNA and defines the nuclear compartment - inner nuclear membrane: contains proteins that act as binding sites for the chromosomes - outer nuclear membrane: composition resembles membrane of ER. continuous with ER membraneNuclear pore: gates where molecules enter or leave nucleus - prevents passage of large molecules but allows small, water-soluble molecules to pass freely and non-selectively between nucleus and cytosol2/22/15 7 Figure 15-8a Essential Cell Biology Drawing of nuclear pore complexes Unstructured protein loops CYTOSOL NUCLEUS Figure 12-10 Molecular Biology of the Cell Small molecules pass freely through nuclear pores small molecules and proteins (up to ~30 kDa) can pass through nuclear pores by free diffusion larger proteins and protein complexes use a receptor system to be transported in and out of the nucleus - protein fibrils protrude from both sides of the pore complex - nuclear side: form a basket like structure. Spacing between the fibrils is wide enough that the fibrils don't obstruct access to the pores2/22/15 8 Figure 15-9 Essential Cell Biology Nuclear transport receptors move molecules through pores (‘NLS’) Figure 15-10 Essential Cell Biology Energy supplied by GTP hydrolysis drives nuclear transport CYTOSOL NUCLEUS - proteins contain a nuclear localization signal that is recognized by nuclear import receptors, which interact with the cytosolic fibrils that extend from the rim of the pore - arrows represent the movement of the receptors - receptors move until nuclear entry triggers cargo release - after cargo delivery, the receptors return to the cytosol via nuclear pores for reusealso see Figure 15-10 in txbk (a little different, visually)- nuclear import receptor picks up a nuclear protein in cytosol and enters nucleus - in nucleus: encounters Ran (which carries GTP) - Ran-GTP binds to import receptor which causes release of nuclear protein - receptor (still carrying Ran-GTP but discharged its cargo in the nucleus) is transported back through the pore to the cytosol - accessory protein triggers Ran to hydrolyze its bound GTP - Ran-GDP falls off import receptor. Receptor is free to bind to another protein2/22/15 9 Figure 15-10 Essential Cell Biology Energy supplied by GTP hydrolysis drives nuclear transport CYTOSOL NUCLEUS Figure 15-10 Essential Cell Biology Energy supplied by GTP hydrolysis drives nuclear transport CYTOSOL NUCLEUS2/22/15 10 Figure 15-10 Essential Cell Biology Energy supplied by GTP hydrolysis drives nuclear transport CYTOSOL NUCLEUS Figure 12-14 Molecular Biology of the Cell Ran, a GTP-binding protein, regulates transport through nuclear pores 95% of Ran is in the nucleus 5% of Ran is in the cytosol GEF = Guanine nucleotide Exchange Factor GAP = GTPase Activating Protein Associated with