Protein Localization Peter Takizawa Department of Cell Biology •Organelles •Distribution of organelles by microtubules and motor proteins •Signal sequences •Examples of protein import 1. Role of signal sequences in targeting proteins. 2. Describe how proteins get into ER, mitochondria, nucleus and peroxisomes. 3. Why. 1. Illustrates important biochemical mechanisms that are common to many biological processes. 2. Classical work in cell biology. 3. Mutations in some pathways lead to disease. Cells contain a variety of organelles that perform specific and unique functions. 1. Each organelle contains unique set of proteins and other components. 2. Organelles perform special and unique set of tasks. 3. Organelles increase efciency by concentrating components of a common pathway in small environment. 4. Protect cell from harmful components. 5. Some organelles surrounded by membrane to prevent loss of material. 6. Should be familiar with function of each organelle. 7. Still image gives impression that organelles randomly distributed. Cells use microtubules to position organelles within their cytoplasm. 1. Cells organize and localize organelles. 1.1. Position where needed. 1.2. Mitochondria at sites of greatest energy consumption. 2. Tether. 3. Transport through cytoplasm. 4. Microtubules play a critical role in organizing cytoplasm of cells. 5. Golgi localize to central region, near nucleus. 6. Disrupting microtubules leads to fragmentation of Golgi and distribution throughout cytoplasm. Microtubules extend throughout the cytoplasm. 1. Microtubules efective for organizing cell because they are long filaments that extend throughout cytoplasm. 2. Large enough to span most cells. 3. Allows cells to position organelles in specific regions. Microtubules are a hollow tube of tubulin dimers. -tubulin + -tubulin - 1. Heterodimer of alpha and beta tubulin. 2. Both bind GTP. 3. Assemble into filaments end on end to form protofilaments. 3.1. 13 protofilaments make a single microtubule. 3.2. Hollow lumen or center. 3.3. Extensive lateral contacts between subunits gives MT greater strength and allows it to grow longer than actin filaments. 3.4. Similar to pvc pipe. 4. Note that microtubules are polarized. 4.1. One end has alpha tubulin and the other end has beta tubulin. 4.2. Grow from their plus ends as dimers associate with plus end. Microtubules grow from their plus ends. Video shows growth of microtubules in vitro. Microtubules grow from their plus ends as more dimers are added to the filament. Occasionally, microtubule will stop growing and then shrink as dimers fall of of plus end. Inside cells, the plus ends of microtubules are capped or stabilized so they don’t shrink. The repeated growing and shrinking allow microtubules to explore diferent areas of the cell and cells can reposition or reorient microtubules. Note that the minus ends of the microtubules emanate from a common center. This region is called the microtubule organizing center and stabilizes the minus ends of microtubules. Proteins link organelles to microtubule network. 1. Cells stabilize minus ends through MTOC. 1.1. Contains large number of proteins -> centrioles. 1.2. Ring of gamma-tubulin that binds and stabilizes minus ends. 2.Location of MTOC determines orientation of MTs. 2.1. In center, MTs radiate outward -> plus ends toward plasma membrane. 2.2. Organelles contain proteins that bind MTs -> serve as tethers. 2.3. Orientation allows cells to control distribution of organelles. 2.4. Kinesins and dynein mediate bidirectional transport of organelles along microtubules. Dynein 1. Motor proteins move along MTs carrying organelles. 2. Motor proteins move in either plus or minus direction. 3. Kinesins large family of motor proteins most of which move toward plus end. 2. Dynein move toward minus end. 3. Motors attach to organelles via receptors in organelle membrane. 4. By controlling binding and activity of motors, cell can dictate movement along MTs and position. Cells regulate activity of motor proteins to control distribution of organelles. Organelles will often have both types of motors on their surface, allowing cells to adjust their position. For example, these melanocytes contain a pigmented organelle called a melanosome. In some organisms, melanocytes position melanosomes in response to the amount of light. In the presence of light. kinesin on the surface of melanosomes moves them to the periphery of cells. In the dark, dynein returns the melanosomes to the center of the cell. Targeting proteins to specific organelles Proteins synthesized in cytoplasm must get to correct organelle. 1. Each organelle contains unique set of proteins and other components. 2. Organelles perform special and unique set of tasks. 3. How do cells get certain proteins to the diferent organelles? 3.1. What targets proteins to these organelles. 3.2. How do they cross the membranes surrounding the organelles? Proteins are imported into organelles from cytosol or transported between organelles. Cytosol Mitochondria Peroxisomes Nucleus Endoplasmic Reticulum Golgi Late Endosome Secretory Vesicles Lysosome Plasma Membrane The synthesis of all proteins starts in the cytosol, but those proteins with signal sequences are delivered to their appropriate organelle. Many organelles receive proteins through the secretory pathway. Proteins are initially translated and inserted into the ER. Proteins are transported to the Golgi and then delivered to the diferent organelles. Some organelles receive proteins directly from the cytosol. The nucleus is a unique organelle as it allows bidirectional movement of proteins. Proteins contain signal sequences that determine their final destination. Function of Signal Sequence Example of Signal Sequence Import into nucleus -Pro-Pro-Lys-Lys-Lys-Lys-Lys-Val- Export from nucleus -Leu-Ala-Leu-Lys-Leu-Ala-Gly-Leu-Asp-Ile + H3N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Glu-Arg-Asn- Import into mitochondria Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu + H3N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala- Import into ER Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln Import into peroxisomes -Ser-Lys-Leu-COO- 1. Proteins contain short sequences that guide them to correct organelle or intracellular destination -> signal sequence. 2. Signal sequence interacts with machinery on organelle that imports protein into organelle. 2.1. How does machinery get to organelle? 3. Signal sequences often found at N-terminus but can be located internally or at C-terminus. 4. Signal sequences often lack exact sequence conservation. 4.1. Can’t identify based on sequence. 4.2. Properties of amino acids that function as signal -> hydrophobic, charged. 5. Glycosylation can also act as targeting signal. Small changes in signal sequences have profound effects on location of proteins. PEXN Human Nucleoside Mitochondria Transporter PARS Mouse Plasma Nucleoside Transporter Membrane Slide illustrates how subtle changes in signal sequences can afect the localization of proteins. In humans, a certain nucleoside transporter contains a 4 amino acid motif that targets it to mitochondria. In mice, the same protein contains two changes in this motif and this targets the protein to the plasma membrane. If you experimentally change the motif in the human protein to the mouse version, the human transporter localizes to the plasma membrane. Is this medically relevant? Fialuridine is a uridine analog that is a potent inhibitor of hepatitis B. Human Mitochondrion Nucleoside Transporter Mouse Mitochondrion In the early 1990’s there was a clinical trial of a drug designed to treat hepatitis B in 15 patients. Hepatitis B is a virus that afects the liver, causing inflammation. One common treatment for this type of virus is nucleoside analogs. Nucleoside analogs are similar to the nucleosides that are normally used to synthesize DNA, so that they can incorporated into DNA, but they are altered so that they stop synthesis or afect the stability of the DNA. Fialuridine is a nucleoside analog that was found to be efective in reducing hepatitis B in animal models. It was subsequently used in human trials and initially it appeared to reduce the amount of virus. But then something went terribly wrong. Long after the clinical trials were concluded, researchers discovered something unique about the way fialuridine afected human cells. Because humans have a certain nucleoside transporter that localizes to mitochondria, fialuridine can enter mitochondria. Why is this bad? Fialuridine poisoned mitochondria, reducing their ability to generate ATP. Why wasn’t this discovered before the clinical trials? Fialuridine was tested on animal models for safety, but those animals were mice and rats whose nucleoside transporter lacks the mitochondrial signal sequence. Consequently, their mitochondria did not take up fialuridine and were not afected by the drug. What happened in the clinical trials? By reducing the activity of mitochondria, fialuridine force energy-intensive cells to start using glycolysis to generate ATP. This lead to an increase in lactate in the blood and a condition called lactic acidosis. Because the liver processes lactate, the increase production of lactate lead to liver failure 7 patients,5 of whom died. Protein import into the endoplasmic reticulum ER signal sequences have a tripartite structure but little sequence similarity. 1. Most proteins that enter ER contains signal sequence at N-terminus. 2. Signal sequence contains 3 domain. 2.1. Most important functionally is hydrophobic sequence. 2.1.1. Will interact with hydrophobic tails of lipids. 2.2. On N-terminal side is series of charge residues of varying length. 2.3. On C-terminal side is set of polar amino acids. 2.3.1. Sequence specifies cleavage by peptidase. 2.3.2. Removes signal sequence from protein. 3. Note lack of homology. Most proteins imported co-translationally into the ER. 1. Most proteins that enter ER do so co-translationally. 1.1. Inserted as they are made. 1.2. Must coordinate translation with insertion. 1.3. Recruit ribosomes to ER membrane. 1.4. Energy consumed during protein translation drives protein through membrane.