Protein Transport in Cells PDF

Group Work Data for the mobility of three di erent proteins (X, Y, and Z) using both uorescent recovery after photobleaching (FRAP) are shown. Separately, single-particle tracking (SPT) data were collected for these samples. PROTEIN Z bleach bleach bleach time ! FRAP fluorescence in bleached...

Group Work Data for the mobility of three di erent proteins (X, Y, and Z) using both uorescent recovery after photobleaching (FRAP) are shown. Separately, single-particle tracking (SPT) data were collected for these samples. PROTEIN Z bleach bleach bleach time ! FRAP fluorescence in bleached area fluorescence in bleached area PROTEIN Y fluorescence in bleached area PROTEIN X time (A) (B) time (C) SPT fl fl ff ff fi ff fi ! A. Assign an SPT pro le (A, B, or C) to each of these proteins on the basis of the respective FRAP pro les. B. It is important to remember that in each of these experiments, the results re ect a real, physical di erence in the way in which these proteins are situated in the plasma membrane. Provide a plausible explanation for the di erences observed in proteins X, Y, and Z. Chapter 11 Review fl ff fl fl ff fl fi ffi fl ff ff • A lipid bilayer has two layers of lipids to form cell membranes. Phosphatidylcholine is very common in the bilayer, having a polar head and two fatty acid hydrophobic tails. Polar head groups on outside, hydrophobic tails on the inside. Amphipathic. • Shorter tails and more double bonds (less saturated) in the tails makes the lipid more uid. Di erence between olive oil and butter where olive oil has more unsaturated fats. Increasing temperature also make fats more uid (butter melts). • Cholesterol is a small molecule with similar amphipathic properties, packs into structure making it more stable. More cholesterol can make membrane more uid. • Membranes generally maintain their orientation. Phospholipids can di use laterally and rotate very quickly, but only go through the membrane to “ ip” after synthesis in ER. This is energetically unfavorable and enzyme that assists, ippase, requires ATP. • Membranes bud o as vesicles and fuse to other membranes, keeping their orientation (cytosolic vs non-cytosolic). • The layer on the outside of cell (the extracellular side) often has glycolipids and glycoproteins that are used in cell-cell communication, protection, etc. • Proteins are embedded throughout, and sometimes span both hydrophilic and hydrophobic areas. Usually alpha helix, but sometimes made of beta sheets called beta barrel. • Proteins function as transporters, anchors, enzymes, and receptors. • Membrane proteins are extremely di cult to purify and study. They are sometimes made soluble in detergents and put into an arti cial bilayer. • Know how FRAP and SPT are used to study rate of di usion in a membrane. Chapter 15 Intracellular Compartments and Protein Transport Emphasis on Membrane-Enclosed Organelles and Protein Sorting Chapter 15 Learning Objectives ff 1. Explain how proteins get into di erent compartments or organelles during translation 2. Explain how signal sequences work to target proteins to organelles 3. How we learned why a protein goes into an organelle or stays in the cytoplasm Where is the telomerase enzyme needed? How does it know how to get there? How proteins get imported Nuclear Pores Proteins remain folded Protein translocators Proteins become unfolded (MT) or are folded after transport (ER) Membrane fusion Proteins remain folded Proteins Carry Signal Sequences f ✴ All proteins are initially made with ATG encoding methionine ✴ Receptors and translocators recognize Signal Sequences ✴ Signal Sequences can be cleaved o Signal sequences ER signal sequence is both necessary and sufficient to direct proteins to ER ER signal sequence is both necessary and sufficient to direct proteins to ER Ways to analyze where protein goes in vitro (How We Know) Common pool of ribosomes no matter where the protein goes Signal sequence on N-terminus of protein being translated dictates whether ribosome goes to rough ER or stays in cytosol What is rough ER? Translation into the ER Lumen 1. ER Signal Sequence is made by ribosome 2. Signal Recognition Particle (SRP) binds to signal sequence and ribosome while protein synthesis pauses 3. SRP receptor guides the ribosome so that it threads the protein being made though protein translocator Synthesis in ER • Soluble proteins have a signal peptide that is cleaved • Soluble proteins are released into the ER lumen • Single-Pass transmembrane proteins have a hydrophobic stop-transfer sequence • Double-Pass have internal hydrophobic start transfer sequence and a hydrophobic stop-transfer sequence, and so on, with more hydrophobic tracts causing multiple-passes. Soluble protein goes into the ER lumen “Single pass” is retained in bilayer “Double pass” has internal ER signal sequence Multiple Choice The gure shows the organization of a protein that normally resides in the plasma membrane. The boxes labeled 1 and 2 represent membrane-spanning sequences and the arrow represents a site of action of signal peptidase. Given this diagram, which of the following statements must be true? fi (a) The N-terminus of this protein is cytoplasmic. (b) The C-terminus of this protein is cytoplasmic. (c) The mature version of this protein will span the membrane twice, with both the N and C-termini in the cytoplasm. (d) None of the above. Group Work fi ff Dr. Outonalimb’s claim to fame is her discovery of forgettin, a protein predominantly made by the pineal gland in human teenagers. The protein causes selective short-term unresponsiveness and memory loss when the auditory system receives statements like “please take out the garbage!” Her hypothesis is that forgettin has a hydrophobic ER signal sequence at its c-terminus that is recognized by an SRP and causes it to be translocated across the ER membrane by the mechanism shown in the gure. She predicts that the protein is secreted from pineal cells into the bloodstream, from where it exerts its devastating systemic e ects. You are a member of the committee deciding whether she should receive a grant for further work on her hypothesis. Fully critique her proposal. Group Work The gure shows the orientation of a multipass transmembrane protein after it has completed its entry into the ER membrane (part A) and after it gets delivered to the plasma membrane (part B). This protein has an N-terminal signal sequence (depicted as the dark gray membrane-spanning box), which signal peptidase cleaves o in the endoplasmic reticulum. The other membrane-spanning domains in the protein are represented as open boxes. Given that any hydrophobic membrane-spanning domain can act as either a start-transfer region or a stop-transfer region, draw the nal consequences of the actions described below on the orientation of the protein in the plasma membrane. Indicate on your drawing the extracellular space, the cytosolic face, and the plasma membrane, as well as the N- and C-termini of the protein. signal peptidase cleavage site ER lumen extracellular space N plasma membrane ER membrane N C cytosol C cytosol (B) (A) ff fi fi fi fi fi A. deleting! the rst signal sequence B. changing the hydrophobic amino acids in the rst, cleaved, sequence to charged amino acids C. changing the hydrophobic residues in every other transmembrane sequence to charged residues, starting with the rst, cleaved, signal sequence Chapter 15 Review • • • • • • ffi • fi • Intracellular compartmentalization involves three mechanisms: 1) transport through large nuclear pores where proteins stay folded, 2) transport across membranes where unfolded proteins are snaked via translocators into e.g. the mitochondria or ER, 3) fusion from one vesicle into another where proteins stay folded. An amino acid “signal sequence” dictates where a protein goes. These certain sequences often have charged or hydrophobic amino acids. Signal sequences are usually necessary and su cient to target a certain organelle. Folded proteins get into the nucleus by a nuclear transport receptor that recognizes positively charged amino acids and through nuclear pore brils. Whether a signal sequence at the amino terminus is present determines if a protein will be made on a free ribosome in the cytosol or will be made on a bound ribosome at the ER. Signal Recognition Particle binds to signal sequence at amino terminus of a nascent protein and ribosome, moving the ribosome to ER and pausing translation. Then the ribosome binds protein translocator and remainder of protein is made while being snaked across ER membrane. Signal peptidase cleaves o a signal peptide at the amino terminus, and if there are no other hydrophobic transfer sequences, the soluble protein goes into the lumen. When a protein is made in the ER and is membrane bound, a sequence of hydrophobic start transfer and hydrophobic stop transfer sequences dictate how many times it will cross the membrane. Scientists follow in vitro whether or not a protein gets into an organelle by 1) centrifuging the mixture and seeing whether it sediments with organelle or free protein or 2) seeing if the protein is protease resistant. Chaperones guide the proper folding of proteins. The Unfolded Protein Response results in the transcription of chaperone genes, makes more chaperone proteins at the ER, and the ER swells. Misfolded proteins can get properly folded. ff •