Exam 1 will be held Friday Oct 2nd in the Tes9ng Center Arjona 110 You must sign up for an available Exam Slot on HuskyCT Exam Registra9on Link Exam slots 11 12 30 12 30 2 2 00 3 30 3 30 5 there is no regular class on exam day There will be 2 Review sessions before the Exam 1 Wednesday 9 30 6 8pm TLS 154 2 Thursday 10 1 6 8pm Arjona 143 Bring your Problem Sets Important The Course Materials link on HuskyCT will be disabled on the exam day BE SURE TO DOWNLOAD ANY MATERIALS YOU WANT FOR LAST MINUTE STUDYING BEFORE FRIDAY Protein Targe9ng get localized to How do proteins di erent places in the cell 1 The Nucleus Organelles in eukaryo9c cells The eukaryoPc cell is characterized by disPnct funcPonal compartments Each compartment is referred to as an organelle A small organ ie like the organs of your body that are Pssues that do a speci c job Nucleus Golgi apparatus Endoplasmic rePculum lysosomes mitochondria chloroplasts etc Each organelle is bounded by at least one membrane Each is de ned in large part by the proteins that are speci cally associated with that organelle There are mul9ple des9na9ons for proteins in eukaryotes There are cytosolic proteins There are extracellular secreted proteins There are plasma membrane proteins There are proteins in the membrane s of each of the organelles There are proteins inside in the lumen of each organelle THE VAST MAJORITY BEGIN TO BE SYNTHESIZED ON CYTOSOLIC RIBOSOMES HOW DO PROTEINS GET WHERE THEY NEED TO GO A roadmap of protein targe9ng in Eukaryotes 1 3 2 4 Topology of Cellular Compartments All protein synthesis starts in the cytoplasm Transport of proteins across organelle membranes can be gated whole folded protein is moved via aqueous pores transmembrane folded or unfolded protein moves via non aqueous transport complexes vesicular protein is transported in small vesicles that fuse with the target compartment Transport of proteins can occur a er the whole protein has been made pos ransla9onally simultaneously with protein synthesis cotransla9onally You will understand the following as we proceed Gated transport is always pos ranslaPonal Transmembrane transport can be post or cotranslaPonal Vesicular transport only occurs a er transmembrane transport PostranslaPonal transmembrane transport usually involves cytosolic chaperones which keep the protein unfolded in the cytoplasm Produc9on of cytosolic proteins is easy to understand mRNA exits nucleus more later Binds free ribosome in cytosol Protein is translated and folds maybe with help of chaperones and or chaperonins ProducPon and targePng of organellar proteins is much more complex How are proteins targeted to speci c places in cells With amino acid sequences that act as address labels No label means cytosolic protein If a protein is made in the cytoplasm and has no targePng signal it will stay in the cytoplasm default localizaPon Non cytosolic proteins contain short sequences of amino acids which act as the targePng signal Speci c labels exist for nucleus mitochondria endoplasmic rePculum etc SomePmes sequenPal combinaPons of labels are used These labels can be conPguous amino acid sequences or combinaPons of mulPple sequences LocaPon of the signal in the protein varies depending on what organelle protein is desPned for These signals are recognized by other proteins that funcPon as receptors and iniPate a sequence of events that lead to the protein being directed to the right place Targe9ng signals can be a linear con9nuous sequence or a patch formed aaer the protein folds Targe9ng signals usually C terminus charged Nuclear Structure Nuclear envelope double membrane surrounding the nucleus Outer membrane is conPnuous with the RER The space between the membranes is equivalent to the outside of the cell interior of ER Think of the membrane as a series of vesicles that are a ened into pancakes and then fused together everywhere but the pores Nuclear pore complex controls entry and exit Nuclear lamina meshwork of intermediate laments lamins under the nuclear envelope Nucleoplasm uid phase of nucleus like cytoplasm ChromaPn DNA protein complex Nucleolus DNA protein complex housing the rRNA genes for producPon of ribosomal RNA Ribosomal subunits assembly Transport into and out of the nucleus Since protein synthesis occurs in the cytoplasm all nuclear proteins are imported Nuclear proteins DNA polymerase histones lamins etc The nucleus breaks down and reforms with each cell division so all nuclear proteins need to be reacquired which is achieved by targePng signals In addiPon many RNAs produced in the nucleus are exported and some reimported with associated proteins mRNAs are transported from nucleus to cytoplasm as mRNPs ribonucleo protein parPcles Ribosomal subunits are assembled in the nucleolus in nucleus and then transported out into the cytoplasm Small nuclear RNAs snRNAs are transported to the cytoplasm assembled with proteins and then transported back into the nucleus as snRNPs that regulate splicing The Nuclear Pore Complex controls entry and exit The Nuclear Pore Complex controls entry and exit NPC forms a large aqueous channel through the double membrane that act as a selecPve barrier to movement in both direcPons A huge complex with a ring basket and plug like structure can see features by EM Made up of about 50 proteins 3000 4000 per nucleus in typical cell 30x the mass of a ribosome Can even trick cell to transport a whole ribosome through it SEM of nuclear membranes Cytoplasmic face Nuclear face TEM of nuclear membrane TEM of puri ed nuclear pores Nuclear Pore Func9on Molecules smaller than 5000 MW move freely through the pore by passive di usion Larger molecules move more slowly by di usion Some up to 40 60 000 depending on size shape and other factors Molecules over 40 60 000 MW 600 amino acids cannot cross freely Average single protein is about 40 000 MW some are 1 000 000 MW Proteins and complexes that are too large to di use are acPvely transported by a process that is not completely understood Import and export occurs via gated transport proteins are transported in fully folded form a er translaPon is complete Nuclear transport is bidirecPonal NPC selecPvity and nuclear targePng signals are what allow di erent proteins to exist in cytoplasm and nucleus Par9cles less than 9 nm in diameter corresponding to a protein with a M W of 40 kD can di use passively through nuclear pores Larger par9cles are transported ac9vely Nuclear transport is bidirec9onal The Nuclear