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LS1a Fall 2014 I. Section Week #9 Membrane transport and membrane potential Small nonpolar molecules (e.g., O2, benzene) and small uncharged but polar molecules (e.g., water) can diffuse through the lipid bilayer without assistance. Large or charged molecules cannot diffuse freely across the membrane (e.g., amino acids, ions, glucose) and require special transport proteins. There are two main types of membrane transport: Passive transport describes the transport of substrates down a concentration and/or electrochemical gradient, such that no additional energy is required. Active transport involves transport of substrates against their concentration and/or electrochemical gradients with the use of energy. The membrane potential of a cell results from a small net imbalance of positive and negative ions on the two sides of the membrane. Potassium leak channels selectively allow potassium ions to diffuse down their concentration gradient out of the cell, leaving behind a net negative charge inside the cell and providing the outside of the cell with a slight positive charge. By allowing potassium ions to flow out of the cell, potassium leak channels are largely responsible for the negative resting membrane potential that exists in most mammalian cells. The resting membrane potential is reached when the concentration and electrical gradients that act on an ion are equal and opposite. The Na+/K+ ATPase pump is responsible for establishing and maintaining the concentration gradient for Na+ and K+ ions across the plasma membrane. It uses energy from ATP hydrolysis to actively transport both ions against their concentration gradients. The pump exports three Na+ ions for every two K+ ions it imports. Section Activity #1: Cells maintain significantly different intracellular and extracellular ion concentrations. a. Are the following ions more concentrated on the inside or outside of the cell? Na+ Higher Inside Higher Outside K+ Higher Inside Higher Outside Cl Higher Inside Higher Outside b. In most mammalian cells, the intracellular concentration of calcium ions (Ca2+) is tightly controlled and maintained constant at about 0.0001 mM. The extracellular Ca2+ concentration in many mammals is close to 2.5 mM. What would be the sign of the membrane potential (inside with respect to outside) in a cell where only Ca2+-selective ion channels were open and all other ion channels were closed? The membrane potential would be positive. Ca2+ ions would move down their concentration gradient to the inside of the cell and leave behind an excess of negative charge outside. Continued on next page… c. Would the sign or magnitude of this potential change if the extracellular Ca2+ concentration were 10 mM instead of 2.5 mM? If so, how it would change? The sign would remain unchanged, but the magnitude would increase. Increasing the concentration of extracellular calcium results in a larger concentration gradient, which 1 c. Would the sign or magnitude of this potential change if the extracellular Ca2+ concentration were 10 mM instead of 2.5 mM? If so, how it would change? The sign would remain unchanged, but the magnitude would increase. Increasing the concentration of extracellular calcium results in a larger concentration gradient, which causes more Ca2+ ions to move inside the cell. This increases the separation of charge across the membrane once the net movement of Ca2+ ions ceases. II. Protein targeting in eukaryotes All proteins are translated by ribosomes that are either freely floating in the cytosol or by ribosomes that are attached to the endoplasmic reticulum (ER). Proteins destined for the endomembrane system (which consists of the ER, the Golgi apparatus, lysosomes, endosomes, and peroxisomes) and for secretion outside the cell must first be translated by a ribosome attached to the ER. Movement between the ER and subsequent compartments in the endomembrane system (or “secretory pathway”) occurs via membrane-bound transport vesicles that are loaded with cargo and subsequently fuse with other compartments. The topology of membrane orientation is conserved throughout the secretory pathway. Signal Sequences are amino-acid sequences within a protein that are necessary and sufficient to target a protein to its correct organelle. The first signal sequence that has to be recognized for a protein to enter the endomembrane system is the ER signal sequence, which is recognized by a protein-RNA complex called the Signal Recognition Particle (“SRP”). Once the SRP binds a ribosome that is translating a protein with an N-terminal ER signal sequence, the SRP halts translation until the ribosome is delivered to the ER membrane. All subsequent signal sequences that determine which organelle a protein will be targeted to are recognized by proteins called “cargo receptors.” Some examples of signal sequences are: Function Sequence + ER signal sequence H3N-MMSFVSLLLVGILPWATEAEQLTKCEVPQ-… ER retention sequence …-(K/H)DEL-COOMitochondrial localization sequence +H3N-MLSLRQSIRFFKPATRTLCSSRYLL-…. Nuclear localization sequence …-PPKKKRKV-… Quality control: the ER prevents proteins that are misfolded or not appropriately oligomerized from leaving. Misfolded proteins may expose amino acid sequences that the ER can recognize causing the protein to be retained until it properly folds. Similarly, proteins that require multiple subunits to properly function can be retained in the ER until all of the appropriate peptide subunits come together. 2 Proteins destined for the nucleus include a “nuclear localization sequence” (an “NLS”) and are synthesized by ribosomes freely floating in the cytosol. Adaptor proteins that float in the cytosol bind to an NLS and shuttle NLS-containing proteins through the nuclear pore into the nucleus. Section Activity #2: Shown below is a simplified diagram of some of the organelles contained inside a eukaryotic cell. The organelles are not drawn to scale. Consider a plasma membrane protein that has been fluorescently labeled so that its location in the cell can be visualized under a microscope. a. Where does translation of this protein take place? In which of the labeled compartments would you expect to observe fluorescence in a cell that is actively translating and expressing this protein? Translation takes place on ribosomes that are attached to the surface of the ER. We would expect to see fluorescence in the ER (specifically in the ER membrane), in the membranes of transport vesicles, in the membranes of the Golgi apparatus (all stacks), and on the cell surface. b. When this membrane protein is present in the endoplasmic reticulum, would its extracellular domain face the cytoplasm or the ER lumen? The ER lumen. c. Use the terms shown below to describe the path that a plasma membrane protein would take from translation to reach its destination. Terms may be used multiple times or not at all. Lysosome Plasma Membrane Vesicles Golgi Apparatus Nucleus Cytosol Endoplasmic Reticulum Peroxisome (Cytosol ) Endoplasmic Reticulum Vesicles Golgi Apparatus Vesicles Plasma Membrane (All ribosomes, even those bound to the ER, are in the cytosol as they translate proteins, which is why it is fine to begin with “Cytosol ER.”) 3 Section Activity #3: You have developed a cell-free (“in vitro”) translation system to study the players involved in the translation of secreted proteins. A series of control experiments are shown below. Microsomes are vesicles derived from ER membranes. Protein X is a known peptide hormone that is secreted from cells. You add the indicated components to the in vitro translation system, incubate for 30 minutes, and then analyze protein production using PAGE, with your results shown below. Lane 1 provides evidence that the in vitro translation system is working as evidenced by the presence of Protein X expression when mRNA is added. a. What are some components that must be included in order to conduct in vitro translation in a test tube rather than in a cell? Ribosomes (large and small subunits, rRNA and ribosomal proteins) Charged tRNAs representing all 20 amino acids (or all 20 tRNAs, all 20 amino acids, and all of the amino-acyl tRNA synthetases, along with ATP) EF-Tu, EF-G, and GTP [Unexpected, but possible answer: Release factors that recognize stop codons and release the synthesized polypeptide] b. Why is the protein in lane 3 so much shorter than in lane 2? When SRP is added, only a small region of Protein X is produced. This suggest that an ER targeting sequence on the N-terminus of Protein X exists and is bound by SRP halting translation until it binds its receptor/the translocon in the ER membrane (or in this case on the microsomes). Since microsomes are absent, translation halts. c. Why are the proteins in lanes 4 and 5 the same size? Where is Protein X located in lane 4 (relative to the ER microsome)? Where is Protein X located in lane 5 (relative to the ER microsome)? Without the SRP to halt translation, the addition of the ER microsomes does not affect the translation of the protein in lane 4, so the protein is fully synthesized outside of the ER microsomes. In lane 5, with both the ER microsomes and the SRP presents, the protein is fully synthesized into the ER microsome lumen. 4 Section Activity #4: Schematic representations of three proteins are shown below. The amino acid sequences highlighted as A-E constitute signal sequences. Identify the function of signal sequences A-E given the results of the mutations described below. a. Deleting the amino acids in region B causes Protein 1 to be secreted. The presence or absence of transmembrane domains determines whether a protein will be embedded in a membrane or not. Proteins that are either secreted or inserted into a membrane via a transmembrane domain must have an ER signal sequence. Unless there is an additional “secondary retention signal,” a protein that enters secretory pathway will either be secreted or end up embedded in the plasma membrane. b. Deleting the amino acids in region D causes Protein 3 to be cytosolic. Deleting the Nt-ER signal sequence from a protein destined for the endomembrane system keeps it in the cytosol. The ER retention sequence does not matter unless the protein is in the endomembrane system where the KDEL receptor is located (which is actually in the cis-Golgi lumen). c. Deleting the amino acids in region B and adding the amino acids of region E to the carboxyterminus of Protein 1 causes it be retained in the ER. Deleting the transmembrane domain causes the protein to be located in the ER lumen, and adding the ER retention sequence at the C-terminus causes the protein to be retained in the ER. d. Deleting the amino acids in region D and adding the amino acids of region C to the aminoterminus of Protein 3 causes it go to the nucleus. Deleting region D causes the protein to be cytosolic, where it can be bound by an adaptor protein and brought into the nucleus once the NLS (region C) is added. The location of where the NLS is added does not matter (as long as it is not buried in the hydrophobic core). e. Deleting the amino acids in region C causes Protein 2 to be cytosolic. Region C must be the nuclear localization signal if removing it causes the protein to be cytosolic 5 Answer: 6