Retrieval of RER enzymes that get into GolgiWhy go backwards (retrograde)?RER proteins that contain a.a sequence KDEL (lys, asp, glu, leu) should stay in RERSome RER enzymes accidentally escape to Golgi in COPII vesiclesThese enzymes can be retrieved by KDEL receptors in COPI vesicles and returned to RERKDEL sequence – recognition key that makes it get retrieved back to the RERCan be altered to be forced to leave or go back to RERLysosome – acidic organelle for degradation (break down things) and recyclingEnergy of ATP hydrolysis is used to pump H+ into the lysosome – acidify the lumen of lysosomeCan pump H+ out to make ATPLysosomal enzyme targeting by Mannose-6-P (targets enzyme to go to lysosome)1. An enzyme destined for the lysosome is phosphorylated on a mannose sugar in the cis golgi. Made in the RER2. Mannose-6-P receptors in trans golgi bind this protein and package it into vesicles3. Clathrin binds to the outside of these vesicles, targeting them to become lysosomes4. Lysosome enzyme is released from the receptor as the lysosome matures5. Clathrin is released and the receptors are recycled to the golgi6. Some mannose-6-p that end up on the plasma membrane are retrieved and recycled. Some fuse with the plasma membraneRole of lysosome in phagocytosis1. Particles are brought in by endocytosis (into the cell)2. The vesicle formed inside the cell is called a phagosome3. Lysosome fuse with phagosomes and transfer their hydrolytic enzymes inside, creating a phagolysosome4. The particle gets digested and nutrients pass into cytoplasm5. The leftover, non-digestible material (garbage) is dumped out from residual bodies by exocytosis (out of the cell)Role of lysosome in autophagy – get rid of dead organelles like mitochondria and chloroplast1. An autophagic vesicle is formed by wrapping ER membrane around a dead mitochondria2. Lysosome fuse with this to make an autophagolysosome3. The lysosome transfer lytic enzymes into this vesicle and the mitochondria is broken down4. The leftovers remain in the residual body until they can be dumped out of the cell by exocytosisno clathrin on the vesiclesReceptor-mediated endocytosis1. Ligand (anything that binds to receptors) bind to receptors on the outside of the cell. A classic example is LDL (low density lipoprotein) binding to LDL receptor. Each receptor has a specific binding to a specific ligand2. Clathrin binds to the transmembrane receptor and endocytotic vesicles form3. The clathrin coat is shed, the endosome is acidified, the ligand is dissociated from the receptor and the rest is sorted into different endosomes and other compartments4. If the ligand is needed by the cell, it is transported to the cytoplasm5. The receptor is recycled to the plasma membrane6. If new receptors are needed, they are made in the RER and trafficked through the endomembrane systemLysosomal fusion with other membranes1. Autophagy: primarily for degredation of dead mitochondriamitochondria + ER -> autophagosome + lysosome -> autophagolysosome2. Phagocytosis: bringing in particles such as bacteria from the outsidephagosome + lysosome -> phagolysosome3. Receptor-mediated endocytosis: ligand binding stimulates endocytosisendocytotic vescicle -> endosome + lysosome -> late endosome (autolysosome)Where have we seen receptors so far?1. Plasma membraneA. facing outside: LDL receptorsB. facing inside: t-SNARES (targets vesicle to bind to plasma membrane)2. Cytoplasm (soluble receptors): importin (NLS), SRP (signal sequence)3. Nuclear membrane: nuclear pore filament (bind importin or NLS)4. RERA. facing cytoplasm: SRP receptor – looks for SRP bound to signal sequenceB. facing lumen: cargo receptors – looks for specific cargo to bind so it can bind the proper coat protein5. GolgiA. facing cytoplasm: transmembrane receptor for COPsB. facing lumen: mannose-6-P receptors6. Transition vesicles: transmembrane receptors and v-SNARES (vesicle)Cellular reproductionTwo main consideration1. Nuclear division – mitosis (not cell division, only prepares for cell division by packing DNA)2. Cell division – cytokinesis (cell movement) – cannot happen without mitosisHow is DNA replicated for mitosis?Meselson-Stahl experiments1. Grow bacteria on “heavy” nitrogen (N-15, radioactive) to metabolically label all newly synthesized DNA2. Then switch to regular “light” nitrogen (N-14, non radioactive). All newly replicated DNA will be “light” but the old, parental DNA will be “heavy” – this way you can tell which DNA is new and which is oldN-15 -> change growth media -> N-143. Use density gradient centrifugation to separate heavy and light DNA. They used cesium density gradients instead of sucroseseparation of bacterial DNA by density centrifugationadd mix of light, heavy and hybrid DNA to cesium density gradientlight on top(natural normal DNA), hybrid on middle, heavy on bottom3 possibilities for replication of DNA in bacteria1. Dispersive: hybrid in the 1st generation – ½ light and ½ heavy2nd gen – same as 1st gen1 band - hybrid2. Conservative: always both heavy and light present, no hybrid2nd gen – either heavy or light, same as 1st gen2 bands – heavy or light3. Semi-conservative: hybrid in the 1st generation but both hybrid and light in the 2nd generation2nd gen – hybrid and light2 bands – hybrid or lightresults of meselson-stahl1. Beginning – all DNA is heavy2. 1st gen – all DNA is hybrid3. 2nd gen – there are hybrid and light bands, very little of the original DNA left so it cant be seenconclusion – bacterial DNA is semi-conservativeReplication of bacterial, circular DNAOriginal strand = parental strand or template strandNew strand = daughter strandSemi-conservativeDNA replication in eukaryotesCant use gradient density centrifugation to separate chromatids or DNA from eukaryotesUse metabolically labeled DNA and look at mitotic chromosomes1. Must use mitotic cells to see compacted chromatids2. Use BrdU (bromodeoxyuridine) to substitute for thymidine in DNAA:T -> A:BrdU3. Thymidine stains dark but BrdU doesn’t4. All T is dark (dominant), all BrdU is light (recessive), hybrid is darklight = all is BrdU (all newly synthesized DNA)dark can be all T or hybridresult : DNA replication is semiconservative1st gen – both chromatids contain 1 strand BrdU and 1 strand thymidine = hybrid, dark2nd gen - one chromatid is dark (hybrid) and the other is light in each mitotic chromosome after 2 generationsmitotic chromosome is half light, half