Sheet, Review Exam 1 By: Brett Schofield WARNING: This is not all of the information that you will need to know, but it is a good guide for your studying. Some of the details have been left out for the sake of brevity. Nuclear Transport: Small proteins (like Ran) and other molecules can diffuse through the Nuclear Pore Complex. Large proteins and protein complexes must go through active transport: o Import NLS containing cargo binds to importin , which binds to importin . This import complex can then enter the nucleus via the Nuclear Pore Complex. Once in the nucleus, Ran-GTP out-competes the NLS containing cargo, and the importin + Ran-GTP complex leaves the nucleus. o Export NES containing cargo binds to exportin, which also cooperatively binds RanGTP. This three-part complex moves to the cytoplasm, where the GTP is hydrolyzed to Ran-GDP, which causes the entire complex to fall apart. Exportin returns alone. Ran-GTP/GDP gradient is formed by the presence of Ran-GEF inside the nucleus (attached to chromosomes), and Ran-GAP in the cytoplasm. Secretory Pathway (vesicle mediated transport): Proteins are co-translationally imported into the ER. They are sorted into buds which are pinched off the ER using the COPII coat complex. These vesicles travel to the Golgi, where they fuse together to form a cis-Golgi. These proteins then travel through the Golgi by cisternal maturation (accomplished by both retrograde [using COPI coated vesicles] and antrograde transport of Golgi resident proteins). From the trans-Golgi, proteins are sorted into clathrincoated vesicles and moved to their final destination (either an organelle or plasma membrane). Vesicles contain a number of factors that allow them to be properly targeted and to mediate membrane fusion. Rab proteins and t- and v-SNAREs aid in targeting. Factors like NSF and SNAP aid in fusion. Cell Signaling: G-Protein Coupled Receptors are 7-pass transmembrane proteins that have GEF activity that is stimulated by association with ligand. This activates a heterotrimeric G protein which splits into or subunits, either or both of which can activate downstream factors. Receptor Tyrosine Kinases are single pass transmembrane proteins that dimerize upon association with ligand. This leads to auto trans-phosphorylation, which recruits Grb2, which recruits Sos (a Ras-GEF). This leads to activation of Ras, which activates Raf (MAPKKK), which activates Mek (MAPKK), which activates Erk (MAPK). MAPK can then translocate to the nucleus, and activate transcription factors. It also has the ability to modulate the activity of various cytoplasmic proteins. Remember that the MAP Kinase proteins are all on a scaffold. Secondary Messengers: Adenylyl Cyclase makes cAMP, which frees PKA from repression. IP3 and DAG start out together, but are split by Phospholipase C. IP3 leads to release of Calcium from ER, which activates Calmodulin-dependent activities. DAG activates PKC. Cytoskeleton: Actin: Exists in both a filament (F-actin) and a monomeric (G-actin) state. In both forms, it has a + (barbed) and (pointed) end. Monomers are preferentially added to the + end of a filament, and preferentially dissociate from the filament at the end, which causes treadmilling. This treadmilling is an essential part of cell mobility as it allows for extension of the lamellapodium.
o Nucleating Factors: ARP2/3 + WASp creates branched filaments, and it is stimulated by the G-proteins Rac (leads to increased lamellapodia formation) and Cdc42 (leads to increased filapodia formation). Formins create linear filaments that bundled get into stress fibers (required for the contraction step of cell mobility). Formins also increase the rate of polymerization by a sliding ring mechanism. It is activated by the Rho G-protein. o Capping proteins: Cap Z (+) and tropomodulin (-) cap the ends of F-actin, and prevent further polymerization or depolymerization. o Other: Profilin binds to end of G-actin, and it exchanges ADP for ATP, thereby recharging actin. Cofilin cleaves F-actin, leading to depolymerization. Thymosin-4 sequesters actin-ATP, lowering the effective concentration of available actin. o Motors: Myosin, which can be either processive or non-processive. Microtubules (MTs): Composed of 13 strands of tubulin dimers (/). Both subunits bind GTP, but only can hydrolyze it. MTs are nucleated at the microtubule organizing center, through the use of -TURC, so only the + end is available for polymerization/depolymerization. The + end shows dynamic instability, where it is constantly growing and shrinking (catastrophe and recovery). This happens by a loss of the GTP-bound cap of tubulin. o Drug effects: Taxol stabilizes, cohchicine destabilizes by increasing GTP hydrolysis. o Katanin cleaves all 13 strands of MTs, often leading to depolymerization. o Motors: Kinesin (usually + end motors) and Dynein ( end motor). Both are usually processive.
Cell Cycle: Interphase: This contains the First and Second Gap phase, in which the cell is growing and making sure that all checkpoints have been reached. It also includes S phase, where the DNA is replicated. M-Phase: Includes Prophase (chromosome condensation), Prometaphase (nuclear envelope breaks down, chromosomes become bi-oriented), Metaphase (chromosomes line up on metaphase plate), Anaphase A (chromosomes pull apart), Anaphase B (spindle poles push apart), and Cytokinesis (splitting of the cytoplasms). Microtubules during Mitosis: increased rate of catastrophe is a result of the presence of destabilizers like kinesin 13, and the absence of key stabilizers like XMAP215. This increased rate of catastrophe allows for the MTs to better search their environment, seeking out the kinetochores on each chromosome. Once found, the kinetochore will stabilize the + end. Spindle formation: contains two poles (usually formed by centromeres, but can be MTs that are bundled together by dynein motors), Astral MTs (MTs growing away from chromosomes), Kinetochore MTs (MTs bound to kinetochores) and Polar MTs (MTs that overlap MTs from the other pole). Sister chromatids are held together by Cohesin until Anaphase. Cyclin Dependent Kinases proteins present throughout the cell cycle, but they are inactive until they are associated with a cyclin (a protein whose levels rise and decline over the cell cycle). In addition, these CDK-cyclin complexes are held in an active, but poised conformation, meaning that they have inhibitory elements (like phosphorylation from Wee1). Maturation/Mitosis Promoting Factor (MPF) is CKD1+Cyclin B. How is Meiosis different? Meiosis is very similar to Mitosis and uses many of the same proteins. However, during Meiosis I, homologous chromosomes must find each other. This is where cross over can occur. These homologous chromosomes them split into two daughter cells, each of which is diploid, but contains two sister chromatids, rather than a single sister chromatid from the maternal and paternal chromosomes. In Meiosis II, the sister chromatids separate forming a total of 4 haploid cells. Remember that in egg formation, usually only 1 viable cell is formed during meiosis the other 3 are discarded.