MCB 2210 1st EditionExam # 2 Study Guide Protein Targeting:- How do proteins get localized to different places in the cell?o The nucleus- Organelles:o The eukaryotic cell contains distinct functional compartments called organelles Each with a specific jobo Bounded by at least one membrane (or more) Defined partially by the proteins that are specifically associated with that organelle- Protein Destinations—cytosol, extracellular (secreted), plasma membrane, organelle membranes, lumens of organelles- Majority of proteins begin to be synthesized on cytosolic ribosomes- All protein synthesis begins in the cytoplasmo Transport of Proteins across organelle membrane can be: Gated: whole folded protein is moved via aqueous pores- Posttranslational transportation  Transmembrane: unfolded protein moves via non-aqueous pores- Both post- and cotranslational transport Vesicular: protein is transported in small vesicles that fuse with the targetcompartment- Only after transmembrane transporto Transport can occur: Posttranslationally: after the whole protein has been made- Ussualy involved cysyolic chaperones—keep the protein unfolded in the cytoplasm Cotranslationally: simultaneously with protein synthesis- Production of Cystolic Proteins:o mRNA exits nucleus binds free ribosome in cytosolprotein is translated and folds- Production and targeting of organelle proteins is more complexo Amino acid sequences act as “address” labels to target specific places in cells No label means it’s a cystolic protein—it will stay in the cytoplasm (default location) Non-cystolic proteins contain short sequences of amino acids which act asthe targeting signal- There are specific labels for each organelle- Sometimes sequential combos of labels are usedThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- These labels can be contiguous sequences or combos of multiple sequences- Location of the signal in the protein varies depending on what organelle the protein is destined foro These signals are recognized by other protein that functionas receptors and initiate a sequence of events that lead to the protein being directed to the right placeo Targeting signals can be a linear continuous sequence or a patch formed after theprotein foldsProtein Targeting in the Nucleus:- Nuclear Structure:o Nuclear Envelope: double membrane around nucleus Outer layer in continuous with the rough ER There is a space between the 2 membrane layers  Series of vesicles that are flatted into pancakes and then fused together everywhere but the pores Nuclear Pore Complex: controls entry and exito Nuclear Lamina: meshwork of intermediate filaments (lamins) under nuclear envelopeo Nucleoplasm: fluid phase of nucleus (like cytoplasm)o Chromatin: DNA/protein complexo Nucleolus: DNA/protein complex that houses the rRNA genes for production of ribosomal RNA (ribosome subunits)- All nuclear proteins are imported since protein synthesis occurs in the CYTOPLASMo Nuclear Proteins: DNA Polymerase, histones, lamins, etc.o The nucleus breaks down and reforms during cell division, so all nuclear proteins need to be reacquired, which is achieved by targeting signals- Many RNAs in the nucleus are exported, some reimported, with associated proteinso mRNA are transported from nucleus to cytoplasm as mRNPs (ribonucleo-protein particles)o Ribosomal subunits are assemble in the nucleolus and then transported out of the cytoplasmo Small nuclear RNAs (snRNAs) are transported to the cytoplasm, assembled with proteins and then transported back into the nucleus- The Nuclear Pore Complex (NPC) controls entry and exit to and from the nucleuso Forms a large aqueous channels through the double membrane that act as a selective barrier to movement in both directions Huge complex with a ring and a plug Made up of about 50 proteins  3000-4000 per nucleus 30x mass of ribosome- Can transport an entire ribosome through ito Molecules smaller than 5000 MW can freely move through pores via passive diffusiono Some up to 40-60,000 MW depending on size and shape, etc.o Molecules over 40-60,000 MW (600 amino acids) can NOT cross freelyo Proteins and complexes that are too larger to diffuse are actively transportedo Import and export occurs via gated transport—proteins are transported in fully-folded form after translation is completeo Transport is BIDIRECTIONALo NPC selectivity and nuclear targeting signals are what allow different proteins to exist in the cytoplasm and the nucleus- Nuclear Localization Signal (NLS): short sequence of amino acids that is the information needed for nuclear importo NECESSARY (you must have to get the protein into the nucleus) and SUFFICIENT (the only signal needed for nuclear targeting) for nuclear targetingo Attach an NLS to the C-terminus of a known cytoplasmic protein Can now be transported to the nucleuso How cells recognize proteins to be transported through the porto Early experiments used the Xenopus oocyte as a model  Very large cells You can easily inject things into the nucleus or into the cytoplasm and seeif they can change compartments Proteins used was nucleoplasmin: pentameric nuclear protein Purify nucleoplasmin Tag protein (fluorescent or radioactive) to see localization Experiment 1:- Inject nucleoplasmin pentamer in oocyte cytoplasm- Protein taken into nuclei- Use proteases to chop protein into head and tail pieces- Experiment 2:o Inject pentameric heads with one tailo Protein taken into nucleus- Experiment 3:o Inject tailo Taken into nucleus- Experiment 4: o Inject pentameric heads into either the cytoplasm or nucleuso No movement- Conclusions:- The whole protein can be taken up into nuclei- The tail is both NECESSARY and SUFFICIENT for uptake (the head is neither)- You can then start at the end of the tail (C-terminus) and keep cutting off amino acids until you get a protein where the tail is not taken up- Defines the NLSo Proteins destined for the nucleus all have a + charged groups of 5-7 amino acids near the C-terminus of the protein- The sequence is not the same for all nuclear proteins, but all are similar and + chargedo Transport is posttranslational and proteins are already foldedo The + charged region must be at the surface of the folded protein so that it can interact with transport proteins or poreo This signal is recognized by