Lipids, Membranes, Transport, and Signaling

Lipids, Membranes, Transport, and Signaling Andy HowardBiochemistry Lectures, Fall 2010 6 October 2010 Lipids, transport, signaling

Lipids, membranes, transfer, and signaling • Lipids are critical components of membranes, where highly selective transfer and signaling processes occur. Lipids, transport, signaling

Lipids, continued Sphingolipids Waxes Steroids Other lipids Membranes Lipid components Fluid mosaic model Lipid rafts Membrane proteins Transport Pores & channels Passive transport Active transport Endo- & exocytosis Signaling G proteins Adenylyl cyclase PIP3 Tyrosine kinases What we’ll discuss Lipids, transport, signaling

Administrative notes • Monday 11 October is fall break—no class • Midterm is Wednesday 13 October • 75 minutes (1:50-3:05) • Same format as midterm #1 • Short lecture follows (3:10-4:05) • Will cover through today’s lecture;normally we stop just short of last lecture, but the holiday allows to include today • No homework due yesterday or today; assignments will be posted for Saturday soon • Answer keys to previous assignments will be posted tonight Lipids, transport, signaling

Sphingolipids • Second-most abundant membrane lipids in eukaryotes • Absent in most bacteria • Backbone is sphingosine:unbranched C18 alcohol • More hydrophobic than phospholipids Lipids, transport, signaling

Varieties of sphingolipids SphingomyelinImage on steve.gb.com • Ceramides • sphingosine at glycerol C3 • Fatty acid linked via amideat glycerol C2 • Sphingomyelins • C2 and C3 as in ceramides • C1 has phosphocholine Lipids, transport, signaling

Cerebrosides • Ceramides with one saccharide unit attached by -glycosidic linkage at C1 of glycerol • Galactocerebrosides common in nervous tissue Lipids, transport, signaling

Gangliosides • Anionic derivs of cerebrosides (NeuNAc) • Provide surface markers for cell recognition and cell-cell communication Lipids, transport, signaling

2-methyl-1,3-butadiene Isoprenoids • Huge percentage of non-fatty-acid-based lipids are built up from isoprene units • Biosynthesis in 5 or 15 carbon building blocks reflects this • Steroids, vitamins, terpenes • Involved in membrane function, signaling, feedback mechanisms, structural roles Lipids, transport, signaling

Steroids • Molecules built up from ~30-carbon four-ring isoprenoid starting structure • Generally highly hydrophobic (1-3 polar groups in a large hydrocarbon); but can be derivatized into emulsifying forms • Cholesterol is basis for many of the others, both conceptually and synthetically Cholesterol:Yes, you need to memorize this structure! Lipids, transport, signaling

Other lipids Image courtesy cyberlipid.org • Waxes • nonpolar esters of long-chain fatty acids and long-chain monohydroxylic alcohols, e.g H3C(CH2)nCOO(CH2)mCH3 • Waterproof, high-melting-point lipids • Eicosanoids • oxygenated derivatives of C20 polyunsaturated fatty acids • Involved in signaling, response to stressors • Non-membrane isoprenoids:vitamins, hormones, terpenes Images Courtesy Oregon State Hort. & Crop Sci. Lipids, transport, signaling

Example of a wax • Oleoyl alcohol esterified to stearate (G&G, fig. 8.15) Lipids, transport, signaling

Isoprene units: how they’re employed in real molecules • Can be linked head-to-tail • … or tail-to-tail Lipids, transport, signaling

Membranes • Fundamental biological mechanism for separating cells and organelles from one another • Highly selective barriers • Based on phospholipid or sphingolipid bilayers • Contain many protein molecules too(50-75% by mass) • Often contain substantial cholesterol too:cf. modeling studies by H.L. Scott Lipids, transport, signaling

Solvent Bilayers • Self-assembling roughly planar structures • Bilayer lipids are fully extended • Aqueous above and below, apolar within Solvent Lipids, transport, signaling

Salmonella ABC transporter MsbAPDB 3B603.7Å2*64 kDa Fluid Mosaic Model • Membrane is dynamic • Protein and lipids diffuse laterally;proteins generally slower than lipids • Some components don’t move as much as the others • Flip-flops much slower than lateral diffusion • Membranes are asymmetric • Newly synthesized components added to inner leaflet • Slow transitions to upper leaflet(helped by flippases) Lipids, transport, signaling

Fluid Mosaic Model depicted Courtesy C.Weaver, Menlo School Lipids, transport, signaling

Physical properties of membranes • Strongly influenced by % saturated fatty acids: lower saturation means more fluidity at low temperatures • Cholesterol percentage matters too:disrupts ordered packing and increases fluidity (mostly) Lipids, transport, signaling

Chemical compositions of membranes (fig. 9.10, G&G) Lipids, transport, signaling

Lipid Rafts • Cholesterol tends to associate with sphingolipids because of their long saturated chains • Typical membrane has blob-like regions rich in cholesterol & sphingolipids surrounded by regions that are primarily phospholipids • The mobility of the cholesterol-rich regions leads to the term lipid raft Lipids, transport, signaling

Significance of lipid rafts:still under discussion • May play a role as regulators • Sphingolipid-cholesterol clusters form in the ER or Golgi and eventually move to the outer leaflet of the plasma membrane • There they can govern protein-protein & protein-lipid interactions • Necessary but insufficient for trafficking • May be involved in anaesthetic functions:Morrow & Parton (2005), Traffic6: 725 Lipids, transport, signaling

Membrane Proteins • Many proteins associate with membranes • But they do it in several ways • Integral membrane proteins:considerable portion of protein is embedded in membrane • Peripheral membrane proteins:polar attachments to integral membrane proteins or polar groups of lipids • Lipid-anchored proteins:protein is covalently attached via a lipid anchor Lipids, transport, signaling

Integral(Transmembrane) Proteins Drawings courtesy U.Texas • Span bilayer completely • May have 1 membrane-spanning segment or several • Often isolated with detergents • 7-transmembrane helical proteinsare very typical (e.g. bacteriorhodopsin) • Beta-barrels with pore down the center: porins Lipids, transport, signaling

Peripheral Membrane proteins • Also called extrinsic proteins • Associate with 1 face of membrane • Associated via H-bonds, salt bridges to polar components of bilayer • Easier to disrupt membrane interaction:salt treatment or pH Chloroflexus auracyanin PDB 1QHQ1.55Å15.4 kDa Lipids, transport, signaling

Lipid-anchored membrane proteins • Protein-lipid covalent bond • Often involves amide or ester bond to phospholipid • Others: cys—S—isoprenoid (prenyl) chain • Glycosyl phosphatidylinositol with glycans Lipids, transport, signaling

N- Myristoylation & S-palmitoylation Lipids, transport, signaling

Membrane Transport • What goes through and what doesn’t? • Nonpolar gases (CO2, O2) diffuse • Hydrophobic molecules and small uncharged molecules mostly pass freely • Charged molecules blocked Lipids, transport, signaling

Transmembrane Traffic:Types of Transport (Table 9.3) Type Protein Saturable Movement Energy Carrier w/substr. Rel.to conc. Input? DiffusionNo No Down No Channels Yes No Down No & pores Passive Yes Yes Down No transport Active Yes Yes UpYes Lipids, transport, signaling

Cartoons of transport types • From accessexcellence.org Lipids, transport, signaling

Thermodynamics ofpassive and active transport • If you think of the transport as a chemical reaction Ain Aout or Aout Ain • It makes sense that the free energy equation would look like this: • Gtransport = RTln([Ain]/[Aout]) • More complex with charges;see eqns. 9.4 through 9.6. Lipids, transport, signaling

Example • Suppose [Aout] = 145 mM, [Ain] = 10 mM,T = body temp = 310K • DGtransport = RT ln[Ain]/[Aout]= 8.325 J mol-1K-1 * 310 K * ln(10/145)= -6.9 kJ mol-1 • So the energies involved are moderate compared to ATP hydrolysis Lipids, transport, signaling

Charged species • Charged species give rise to a factor that looks at charge difference as well as chemical potential (~concentration) difference • Most cells export cations so the inside of the cell is usually negatively charged relative to the outside Lipids, transport, signaling

Quantitative treatment of charge differences • Membrane potential (in volts  J/coul):DY = Yin - Yout • Gibbs free energy associated with difference in electrical potential isDGe = zFDYwhere z is the charge being transported and F is Faraday’s constant, 96485 JV-1mol-1 • Faraday’s constant is a fancy name for 1. Lipids, transport, signaling

Faraday’s constant • Relating energy per moleto energy per coulomb: • Energy per mole of charges,e.g. 1 J mol-1, is1 J / (6.022*1023 charges) • Energy per coulomb, e.g, 1 V = 1 J coul-1, is1 J / (6.241*1018 charges) • 1 V / (J mol-1) =(1/(6.241*1018)) / (1/(6.022*1023) = 96485 • So F = 96485 J V-1mol-1 Lipids, transport, signaling

Total free energy change • Typically we have both a chemical potential difference and an electrical potential difference so • DGtransport = RTln([Ain]/[Aout]) + zFDY • Sometimes these two effects are opposite in sign, but not always Lipids, transport, signaling

Pores and channels • Transmembrane proteins with centralpassage for small molecules,possibly charged, to pass through • Bacterial: pore. Usually only weakly selective • Eukaryote: channel. Highly selective. • This is an oversimplification: there are bacterial channels too • Usually the DGtransport is negative so they don’t require external energy sources • Gated channels: • Passage can be switched on • Highly selective, e.g. v(K+) >> v(Na+) Rod MacKinnon Lipids, transport, signaling

Protein-facilitated passive transport • All involve negative DGtransport • Uniport: 1 solute across • Symport: 2 solutes, same direction • Antiport: 2 solutes, opposite directions • Proteins that facilitate this are like enzymes in that they speed up reactions that would take place slowly anyhow • These proteins can be inhibited, reversibly or irreversibly Diagram courtesy Saint-Boniface U. Lipids, transport, signaling

Kinetics of passive transport • Michaelis-Menten saturation kinetics:v0 = Vmax[S]out/(Ktr + [S]out) • Vmax is velocity achieved with fully saturated transporter • Ktr is analogous to Michaelis constant:it’s the [S]out value for which half-maximal velocity is achieved. Lipids, transport, signaling

Lineweaver-Burk plot for transport Vmax = 0.5 mMs-1 Ktr = 0.1 mM Lipids, transport, signaling

Primary active transport • Energy source is usually ATP or light • Energy source directly contributes to overcoming concentration gradient • Bacteriorhodopsin: light energy used to drive protons against concentration and charge gradient to enable ATP production • P-glycoprotein: ATP-driven active transport of many nasties out of the cell Lipids, transport, signaling

Secondary active transport • Active transport of one solute is coupled to passive transport of another • Net energetics is (just barely) favorable • Generally involves antiport • Bacterial lactose influx driven by proton efflux • Sodium gradient often used in animals Lipids, transport, signaling

Complex case: Na+/K+ pump • Typically [Kin] = 140mM, [Kout] = 5mM,[Nain] = 10 mM, [Naout] = 145mM. • ATP-driven transporter:3 Na+ out for 2 K+ inper molecule of ATP hydrolyzed • 3Na out: 3*6.9 kJmol-1,2K in: 2*8.6 kJmol-1= 37.9 kJ mol-1 needed, ~ one ATP Diagram courtesy Steve Cook Lipids, transport, signaling

What’s this used for? • Sodium gets pumped back in in symport with glucose, driving uphill glucose transport • That’s a separate passive transport protein called GluT1 Diagram courtesy Steve Cook Lipids, transport, signaling

How do we transport big molecules? • Proteins and other big molecules often internalized or secreted by endocytosis or exocytosis • Special types of lipid vesicles created for transport Lipids, transport, signaling

Receptor-mediated endocytosis • Bind macromolecule to specific receptor in plasma membrane • Membrane invaginates, forming a vesicle surrounding the bound molecules (still on the outside) • Vesicle fuses with endosome and a lysozome • Inside the lysozyome, the foreign material and the receptor get degraded • … or ligand or receptor or both get recycled Lipids, transport, signaling

Example: LDL-cholesterol Diagram courtesyGwen Childs, U.Arkansas for Medical Sciences Lipids, transport, signaling

Exocytosis Diagram courtesy LinkPublishing.com • Materials to be secreted are enclosed in vesicles by the Golgi apparatus • Vesicles fuse with plasma membrane • Contents released into extracellular space Lipids, transport, signaling

Transducing signals • Plasma membranes contain proteins called receptors that allow the cell to respond to chemical stimuli that can’t cross the membrane • Example: Bacteria can detect chemicals.if something useful comes along,a signal is passed from the receptor to the flagella, enabling the bacterium to swim toward the source Lipids, transport, signaling

Multicellular signaling • Hormones, neurotransmitters, growth factors all can travel to target cells and produce receptor signals Diagram courtesy Science Creative Quarterly, U. British Columbia Lipids, transport, signaling

Extracellular signals • Internal behavior ofcells modulated byexternal influences • Extracellular signals are called first messengers • 7-helical transmembrane proteins with characteristic receptor sites on extracellular side are common, but they’re not the only receptors Image courtesy CSU Channel Islands Lipids, transport, signaling

Lipids, Membranes, Transport, and Signaling