Ion Distribution Gives Rise to the Membrane Potential

Transcription

1 Ion Distribution Gives Rise to the Membrane Potential Ion flow across the cell membrane is detected as an electric current, and an accumulation of ions, if not perfectly balanced by an accumulation of oppositely charged ions, is detectable as an accumulation of electric charge, or membrane potential. The membrane potential comes from a thin (<1 nm) layer of ions close to the membrane, held in place by electrical interaction with their oppositely charged counterparts on the other side of the membrane. nly a tiny fraction of the ions in the cell must move across the membrane to create a membrane potential. In animal cells, there is a slight negative charge inside of the cell membrane and a slight positive charge outside. Therefore, the membrane potential favors the entry of cations (positively charged) and disfavors entry of anions (negatively charged) into the cell. 1

2 Generating the Membrane Potential The membrane potential in animal cells comes largely from ion flow generated by the potassium gradient across the membrane. The high intracellular potassium concentration inside cells is generated by the sodium-potassium pump, which actively pumps potassium into the cell and pumps out sodium. Potassium inside the cell is electrically balanced by interaction with a number of organic anions. The membrane also contains potassium leak channels, which flicker between open and closed states no matter what the conditions are inside or outside the cell (they are not gated). When they are open, potassium ions flow freely down the steep concentration gradient out of the cell; the movement of positively charged ions out of the cell leaves behind an excess of negative charge (the organic anions cannot leave the cell) and creates an electric field, or membrane potential. This membrane potential will oppose the further movement of potassium out of the cell. Within milliseconds an equilibrium condition is established in which the membrane potential is just strong enough to counterbalance the tendency of potassium to move down its concentration gradient - that is, in which the electrochemical gradient for potassium is zero, even though there is a much higher concentration of potassium inside the cell than out. This potential is referred to as the resting potential of the cell. The resting potential can be calculated from the relative difference in concentration of the ion inside and outside the cell (larger concentration differences lead to larger membrane potentials). If you go on to study neuroscience you will see how the interplay between membrane potential and ion channels is used for electrical signaling in nerve cells. Extra information on the sodium-potassium pump: This pump is an active transporter that uses energy from ATP hydrolysis to pump potassium and sodium against their electrochemical gradients. For every 3 sodium ions it pumps out of the cell, it pumps 2 potassium ions in. This creates a charge imbalance which contributes to the membrane potential. owever, this effect accounts for only ~10% of the membrane potential. Most of the membrane potential comes from the potassium gradient and potassium ions moving out through leak channels. 2

3 Summary of Main Points Proteins of different functions associate in different ways with membranes; regions of proteins that are contained within the bilayer are hydrophobic The membrane imposes a barrier to diffusion of large, polar and all charged molecules; these molecules must be moved across membranes through transport proteins The inside and outside of the cell have very different ion compositions that are maintained by transporters and channels Electrochemical gradient influences how ions will move across the membrane Ion channels transport ions with tremendous selectivity and efficiency; structural features of the channel enable them to do so Membrane potential originates from a charge imbalance due to the movement of potassium ions 3

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5 Membrane Fusion, Cell Structure and Intracellular Trafficking 1. IV entry into host cells a. IV-host cell membrane fusion b. Fuzeon and inhibition of membrane fusion 2. verview of structure of prokaryotic and eukaryotic cells a. Advantages and challenges of compartmentalization 3. Transport into and out of organelles a. Studying trafficking using GFP fusion proteins b. Signal sequences c. Transport into the ER 4. Functions of the ER a. Protein glycosylation 5. Secretory pathway a. Vesicular transport of proteins and lipids b. Coated vesicles Lecture Readings Alberts ; McMurry

6 IV Entry into ost Cell IV ~1.5 nm host cell host cell ow that we know more about the properties of membranes, let s go back to the issue of viral entry into the cell. The virus enters cells by fusing its membrane with the plasma membrane, allowing the viral genome to enter the cell. Membrane fusion is energetically unfavorable and does not occur spontaneously. Why? In order to fuse, two membranes must be brought into close apposition (~1.5nm apart), squeezing out water and bringing the charged lipid head groups close together. The juxtaposition of charged head groups results in unfavorable electrostatic interactions. This is similar to an example David told you about earlier in the course - the association of two charged DA strands of a double helix, which must overcome unfavorable charge-charge repulsion. ow are the viral and host cell membranes brought so close together when their juxtaposition is energetically unfavorable? 6

7 verview of Viral-ost Cell Membrane Fusion viral membrane IV CD4 gp41 gp120 gp41 C-term gp41 cell membrane chemokine receptor gp41 -term fusion peptide FREE VIRUS CD4 ATTACMET CEMKIE RECEPTR BIDIG MEMBRAE ISERTI MEMBRAE FUSI The same proteins that are involved in virus-host cell recognition play a role in membrane fusion. We discussed how IV Gp120 binds host cell CD4 protein first (CD4 ATTACMET), and this binding changes Gp120 so that it recognizes the chemokine co-receptor (CEMKIE RECEPTR BIDIG). Binding of Gp120 to the chemokine receptor triggers another change in Gp41 which reorganizes an amino-terminal helical region (blue-green) and attached fusion peptide that was previously buried in the unbound structure, allowing Gp41 to insert into the membrane of the target cell (MEMBRAE ISERTI). The fusion peptide is extremely hydrophobic; this is why it is stable either buried in the hydrophobic core of the folded Gp41 protein, or in the hydrophobic interior of the bilayer. The fusion protein Gp41, which really consists of three identical subunits of Gp41 (when 3 protein chains come together it is referred to as a trimer), thus becomes transiently anchored in two opposing membranes. ow does this cause membrane fusion? The fusion protein Gp41 spontaneously rearranges - it changes its structure - into a very stable, tightly packed bundle of alpha-helices. The energy released by this favorable structural rearrangement is used to pull the two membranes together, overcoming the unfavorable electrostatic repulsion of charged lipid head groups that normally blocks membrane fusion (MEMBRAE FUSI). Therefore, the fusion protein Gp41 is similar to a mouse trap - the IV fusion protein contains a reservoir of potential energy which is released and harnessed to do the work of bringing two membranes together. 7

8 Viral-ost Cell Membrane Fusion Animation of IV-host cell fusion 8

9 Steps in Viral-ost Cell Membrane Fusion cell membrane fusion peptide gp120 CD4 co-receptor gp41 -term slow gp41 gp41 C-term viral membrane ATIVE PRE-AIRPI ITERMEDIATE AIRPI FUSI PST- FUSI The process of IV-host cell fusion has been well studied using biophysical and structural methods. These experiments have provided insight into the structure of intermediates in this process and the relative timescales for conversion of one state into another. Specifically, as we mentioned, CD4 and co-receptor binding to gp120 triggers a change in gp41 that orders the amino-terminal region (-term; yellow-green) into a helix and frees the aminoterminal fusion peptide, allowing it to insert into the plasma membrane of the host cell; this intermediate is called the pre-hairpin intermediate. Conversion of the pre-hairpin intermediate to the hairpin, where the IV and host cell membranes are closely juxtaposed and gp120 has dissociated, is a slow process; this slow kinetic conversion is crucial in the efficacy of fusion inhibitors used in IV treatment. Ultimately, the hairpin is converted into the fused state in which mixing of lipids between the two bilayers takes place. 9

10 Energy Diagram for gp41 Conformational Change We can use an energy diagram to represent the energetics of the structural change in gp41. You will see these diagrams again introduced in much more depth when David lectures. n the x-axis we are plotting the progress of the reaction (also referred to as the reaction coordinate) - this is the series of states that gp41 goes through along the way from native gp41 (complexed to gp120 but in the absence of CD4 or co-receptor) to the hairpin state. n the y- axis we are plotting free energy (G). The energy of the hairpin state is lower than that of the native state of gp41 (solid black line); therefore, the ΔG for this reaction is negative. This means that when gp41 is made and folds it does not fold to the lowest free energy state! As you heard from Dan, ΔG tells us about whether a reaction will occur spontaneously or not (thermodynamics). egative ΔG tells us that gp41 spontaneously converts to the hairpin state, but it does not tell us anything about how fast the reaction will occur. In this case, there is a barrier (activation energy) separating native gp41 from the hairpin state; this barrier makes the reaction very very slow. This is good because the virus does not want native gp41 to convert to the hairpin in the absence of CD4 and the co-receptors. What tells us how fast this reaction will go (kinetics) is the activation energy - the difference in energy between the reactants (gp41) and the highest energy state in the progress of the reaction (transition state). To reach the transition state bonds must be broken (unfavorable enthalpy) and molecules must interact and collide to react productively (unfavorable entropy). The larger the activation energy, the slower the rate of the reaction. ative gp41 does not convert to the hairpin state on any reasonable timescale (in the absence of CD4 and coreceptor) because the activation energy is high. What CD4 and co-receptors are thought to do (based on studies of influenza fusion proteins) is to destabilize the native state, raising its free energy, lowering the activation energy, and therefore making the reaction go faster (red line). 10

11 2 Asn 2 Ser Glu Lys 3 Asn 2 Glu Glu Gln 2 Gln Fuzeon: IV Fusion Inhibitor Leu Glu Glu Glu Ile Leu Asp Ser is 3 Ile Leu Ser Ser Leu Leu Lys Leu Ala Trp Trp Thr Tyr 2 2 Asn Trp Phe Fuzeon is a clinically approved IV fusion inhibitor that blocks fusion of the viral and host cell membranes. Fuzeon is a 36 amino peptide corresponding to a region from the carboxylterminus of Gp41. Fuzeon was not designed to be a fusion inhibitor - it was discovered as part of an effort to develop a vaccine against IV (targeted against Gp41). Scientists serendipitously discovered that peptides from the Gp41 fusion protein had an antiviral effect when incubated with T cells. At the time they did not understand why the peptides had this activity, but as more was learned about the process of membrane fusion, scientists were able to formulate a hypothesis. Extra information on vaccine development: Vaccines are typically developed by immunizing animals with either whole, killed viruses or killed bacteria (these are referred to as attenuated because the viruses and bacteria used for immunization are not capable of causing an infection), or by immunizing with a protein or fragment of a protein derived from the pathogen (virus or bacterium). In each case the immune system develops antibodies against these foreign molecules. The antibodies are then involved in recognizing and helping to eliminate the pathogen when the host is presented with it again. Many strategies have been attempted to produce a vaccine for IV, including immunization with the coat proteins gp41 and gp120. one of these strategies has been effective, primarily because the virus constantly generates mutants that differ in the sequence of gp41, gp120 and other viral proteins. Although the high IV mutation rate produces many non-functional viruses, it produces some functional, infectious ones that are able to avoid detection and killing by the immune system. 11

12 Inhibition of Membrane Fusion by Fuzeon cell membrane AIRPI FUSI PST- FUSI gp120 -term CD4 co-receptor slow gp41 C-term C-peptide (Fuzeon) viral membrane IIBITED ITERMEDIATE ATIVE PRE-AIRPI ITERMEDIATE ow does Fuzeon inhibit the membrane fusion process? Fuzeon inhibits membrane fusion by acting as a decoy that sequesters the amino-terminus (term) of gp41 so that it is unavailable to bind to the authentic carboxy-terminal region (C-term) of the protein. The Fuzeon peptide (C-peptide), which corresponds to a sequence found in the carboxyl-terminus of Gp41, binds tightly and specifically to the pre-hairpin intermediate. Binding of Fuzeon prevents the conversion of the pre-hairpin intermediate to the hairpin state which holds the membranes close enough for membrane fusion and lipid mixing to occur. Therefore, Fuzeon binds to the pre-hairpin and converts it to an inhibited intermediate that is not capable of rearranging to the hairpin state (as long as the Fuzeon peptide remains bound). Fuzeon binds to Gp41 only after Gp120 interacts with its cellular receptors; this is one piece of experimental evidence that Fuzeon acts on the pre-hairpin intermediate. 12

13 Inhibition of Membrane Fusion by Fuzeon Fuzeon inhibition of IV-host cell fusion 13

14 IV Life Cycle recognition of host cell viral amplification/ replication IV entry into cell viral assembly and exit from cell host cell ow that we understand the first two steps in the viral life cycle - how the virus recognizes its host cells and then enters them by fusing its membrane with that of the host cell - let s turn to the last two steps in the life cycle. nce the virus gets its genome into the host cell, it must make more copies of its genome and associated structural proteins (step 3). We will not talk about this step now - Rob will tell you about this in the next part of the course. After the viral genome is copied and associates with structural proteins, the virus must assemble into a functional form containing a membrane and glycoprotein coat and then exit the cell (step 4). ow does virus get the coat and leave cell? To understand the answer to this question, we need to know more about the structure of eukaryotic cells. 14

15 Cell Architecture >10 µm This slide highlights the differences in structure between prokaryotic and eukaryotic cells. Prokaryotic Cells There are no internal compartments or organelles in prokaryotic cells, such as bacteria. The genetic material is contained within the cytoplasm. Bacterial cells are surrounded by a plasma membrane and a cell wall, composed of proteins and carbohydrates, which provides additional rigidity and protection for the cell. In between the plasma membrane and the cell wall is the periplasmic space. Some bacteria have a flagellum that allows the cell to move. Eukaryotic Cells Eukaryotic cells are generally larger and more elaborate than prokaryotes. Some live as singlecelled organisms whereas others live in assemblies of many cells. All eukaryotic cells have a nucleus where the genetic material is stored and where DA is copied into RA (the process of transcription). Recall that RA is converted into proteins by translation on ribosomes, a process that occurs in the cytoplasm (also called cytosol). Cells have a number of organelles in the cytoplasm, each of which is surrounded by a membrane. These include mitochondria, which are the energy generators of the cell - they harness energy from food to make ATP - the chemical that fuels cell activities. The cytoplasm also contains endoplasmic reticulum (ER), a maze of of interconnected spaces surrounded by a membrane, which serves as the site of synthesis of proteins destined for membranes. The Golgi apparatus is a stack of flattened disks of membrane that receives proteins from the ER, modifies them and directs them to other organelles, the plasma membrane or to the exterior of the cell (carried in secretory vesicles). Lysosomes are sites of degradation of macromolecules (a sort of trash can), and peroxisomes are a contained environment for reactions involving hydrogen peroxide, a highly reactive molecule. Like prokaryotic cells, the cytoplasm is surrounded by the plasma membrane. All organelles are surrounded by a single membrane bilayer, with the exception of the nucleus and mitochondria. The nucleus is surrounded by a double membrane bilayer (called the nuclear envelope) that is contiguous with the ER and mitochondria also have a two membrane structure comprised of inner and outer bilayers. 15

16 Compartments Provide Advantages to Cells: Specialized environments But Also Challenges: ow do proteins move across membranes between compartments? ow are proteins targeted to the correct compartments? The striking difference between prokaryotic and eukaryotic cells is the abundance of intracellular compartments in eukaryotes. Since bacteria have clearly been evolutionarily successful (they account for a significant fraction of the biomass on earth and there are a large number of species), what is the advantage of having compartments? In short, compartments allow for separation of activities and for specialized environments. For example, as we will learn, the ER has an environment similar to that outside the cell. The ER is an oxidizing environment, in contrast with the cytosol, which is reducing. The oxidizing environment of the ER favors disulfide bond formation, which would not be favored in the cytosol. Although compartments provide advantages to cells, they also create new challenges. Specifically, the barrier imposed by membranes surrounding compartments means that cells have had to evolve mechanisms to move proteins across these membranes. Additionally, cells have had to evolve mechanisms to target the correct set of proteins to the correct compartments. We will discuss how the cell deals with these challenges in the next few slides. 16

17 Transport Into and ut of rganelles nuclear pore nucleus transport through nuclear pores chloroplast proteins transport across membranes mitochondrion ER vesicle protein in plasma membrane ribosomes Golgi transport by vesicles Proteins are imported into organelles by three different mechanisms: (1) Proteins and nucleic acids move into (and out of) the nucleus through channels in the membrane called nuclear pores. These are protein-lined structures that span both the inner and outer membranes of the nuclear envelope. uclear pores are large enough that ions and small molecules (e.g. metabolites) can freely diffuse through them, but proteins and nucleic acids cannot. Rob will tell you much more about the process of nuclear transport in the next set of lectures. Again, note that the membranes of the ER are contiguous with the nuclear envelope. (2) Proteins move into chloroplasts (plant organelles that are involved in photosynthesis, the process of harvesting light into energy for the cell), ER and mitochondria by being transported across the membranes of these organelles. Unlike nuclear pores, there exist no channels in the membrane of mitochondria through which ions or metabolites can move. (3) Proteins move within the secretory pathway - between the ER, Golgi, plasma membrane, and lysosomes - inside lipid vesicles. As we will see, vesicles are loaded with cargo proteins from the lumen, or interior space, of one compartment, and discharge their cargo into a second compartment. We will talk about an example of transport by vesicles, by discussing how proteins traffic from the ER to the Golgi and on to the plasma membrane. As you will see, all of this information is critical for understanding how IV co-opts the host cell to produce functional viral particles during an infection. The processes we will discuss next are important for generating functional, glycosylated gp41 and gp120, without which the virus could not gain entry into host cells. 17

18 mutant viral coat protein-gfp fusion ow do we know how proteins traffic within cells? Protein-GFP fusions can be used to visualize trafficking of proteins in single, living cells, in real time. These kinds of experiments help scientists know how proteins move within cells. In this experiment, scientists studied trafficking of a viral coat protein fused to GFP, a protein that is synthesized on the surface of the ER and then travels through the secretory pathway to the plasma membrane. This viral coat protein is a mutant version that is only trafficked within cells at low temperature. The cells expressing the viral coat protein-gfp fusion were kept at high temperature so that the fusion protein was synthesized and localized to the ER, but was not trafficking through the cell. When the temperature was lowered, the viral coat protein-gfp fusion was transported from the ER to the Golgi, and then finally to the plasma membrane. This kind of experiment helps scientists know how proteins move from the ER to the plasma membrane within cells. The path taken by this viral coat protein is identical to the path taken by the IV coat proteins we will talk more about. 18

19 Targeting Proteins to the Correct Destination: Signal Sequences Let s now turn to the issue of how proteins are targeted to the correct compartment. Signal sequences, stretches of amino acids within protein sequences, play a crucial role in targeting proteins to their correct destinations within cells. Typical sorting signal on proteins (acting like a zip code to specify the correct location) can either be contiguous stretches of amino acids or they can consist of amino acids distributed throughout the protein sequence, which are close together in the folded structure of the protein. Signal sequences are recognized by transport receptors (at least one transport receptor exists for each compartment) which are involved in targeting proteins to a compartment (typically because the receptor protein binds a another protein on the compartment that is unique to the compartment and specifies the compartment identity). 19

20 Role of Signal Sequences in Protein Targeting For example, ER signal sequences are used to target proteins to the ER. Proteins that contain an ER signal sequence are targeted to that compartment, whereas those that do not have one remain in the cytosol. ow do we know this? Signal sequences are necessary and sufficient to direct a protein to a particular organelle. What does this mean? ecessary - If you remove the ER signal sequence, the protein no longer goes to the ER. Sufficient - If you attach an ER signal sequence to a protein that did not have one (and was normally cytosolic), it is targeted to the ER. 20

21 Targeting of Proteins to the ER ER ribosomes lysosome protein in plasma membrane Golgi The ER is the most extensive membrane system in the cell. Unlike other organelles, it serves as the entry point for proteins destined for other organelles, as well as the ER. Proteins that are targeted to the lysosome, the Golgi, and the plasma membrane all enter the ER first. nce inside the ER, proteins generally do not re-enter the cytosol. Two kinds of proteins are transferred from the cytosol to the ER: (1) Water-soluble proteins that will be moved to the ER lumen. (2) Transmembrane proteins that are partially translocated across the ER membrane and end up embedded in it. Both kinds of proteins are directed to the ER by signal sequences. Unlike proteins that enter other organelles, most proteins that enter the ER do so before they are completely synthesized. We will not talk in detail here about how proteins are moved across the ER membrane into the lumen of the ER. owever, one unique aspect of this process is that it generally occurs co-translationally - as the polypeptide is being made it gets moved across the ER membrane through a special channel. 21

22 Glycosylation of Proteins Begins in the ER X Y X = sidechains cannot be Pro or Asp Y = sidechains are Ser or Thr monosaccharide oligosaccharide C 2 1 mannose Most proteins entering the ER are covalently modified there. Disulfide bonds are formed between pairs of cysteine sidechains (you heard about these from Dan); this is enabled by the specialized oxidizing environment of ER (that is different from the cytosol, which is reducing and would therefore not favor disulfide bond formation). Glycosylation is the covalent modification of proteins with carbohydrate, or sugar, molecules. The most common way carbohydrate groups are attached to proteins is through linkage to the amide (2) group of an asparagine side chain - these are called -linked oligosaccharides. Glycosylation of proteins begins in the ER (e.g. for glycoproteins like Gp41 and Gp120) with attachment of a preassembled oligosaccharide (composed of monosaccharides, single sugar subunits) to an asparagine side chain found within a specific amino acid sequence on the protein (Asn-X-Ser or Asn-X-Thr). This short sequence is recognized by an oligosaccharyl transferase that resides in the ER membrane. Further processing (trimming to remove some of the monosaccharide subunits and to add others) of the oligosaccharide occurs in the ER and then is finally completed in the Golgi. The monosaccharides that modify proteins are hexoses (they have six carbon atoms). Ribose, which you have heard about in nucleic acids, is a pentose (5 carbon atoms). These molecules are chiral and most sugars belong to the D family (unlike the naturally-occurring amino acids which are L). umbering of the ring positions begins with the carbon indicated and proceeds clockwise. Glycoproteins have important functions at the surfaces of cells. The protein portion of the glycoprotein lies within the cell membrane and the carbohydrate portion extends into the extracellular environment. ligosaccharides can function as receptors for other cells (proteins in the surface of these cells may bind to the oligosaccharide; an example of this is Gp41 and Gp120), microorganisms, or for drugs. Glycoproteins are also responsible for the differences between different blood types - different red blood cells from people with different blood types display different oligosaccharides. 22