Cellular Biochemistry Fall Semester 2013 Sept. 23 Benoit Kornmann Institute of Biochemistry
Introduction to biological membranes General functions and properties Membrane lipids Physical properties Distribution/asymmetry Membrane proteins Integral proteins Lipid-anchored proteins Peripheral proteins Distribution and signaling Phosphoinositides (Vesicular transport) Transporter Main textbook Molecular Biology of the Cell 5th edition Alberts et al. Garlands eds. 2
c Function of the biological membranes Allow COMPARTIMENTALIZATION Plasma membrane defines the boundary of the cell Intracellular membranes subdivide the cell into different compartments (organelles) that differ in composition (proteins, ions, ph, redox potential, electrochemical potential) These compartments carry out different cellular functions Favor biochemical reactions 2D instead of 3D diffusion favors encounter of metabolites 3
Biological membranes 200 ms in the life of aquaporin (B.L. de Groot and H. Grubmüller: Science 294, 2353-2357 (2001) From BM/0C www.mpibpc.mpg.de/grou ps/de_groot/gallery.html Media 10.1 Membranes are thin (5-7 nm), strong, non-elastic, self-sealing, flexible, deformable, hydrophobic barriers between aqueous compartments Composed of amphiphilic lipids (phospholipids) arranged as a bilayer. The bilayers also contain non-membrane-forming lipids, and proteins many of which carry carbohydrate moieties or covalently bound lipids. The bilayer is usually fluid, mostly liquid 4
Challenges in membrane biology Membranes derive from membranes membranes inherited from sperm and oocyte how to make new membranes? Growth: one molecule at a time (lipid/protein) Fusion/fission How to make the right amount of membrane? How can the cell differentiate between two membranes? E.g. what differs between an early and late endosome, how is that difference generated and maintained? How can a protein be targeted to the right membrane? Limited permeability How to selectively transport metabolites and nutrients when needed? How to communicate across membranes? 5
Introduction to biological membranes General functions and properties Membrane lipids Physical properties Distribution/asymmetry Membrane proteins Integral proteins Lipid-anchored proteins Peripheral proteins Membrane Identity Phosphoinositides Microdomains Exemples 6
Glycero-phospholipids 7
Glycero-phospholipids Figure 10-3 Molecular Biology of the Cell ( Garland Science 2008) 8
Glycero-phospholipids Figure 10-3 Molecular Biology of the Cell ( Garland Science 2008) 9
Sphingolipids Glycolipids Figure 10-3 Molecular Biology of the Cell ( Garland Science 2008) 10
sterols Figure 10-4 Molecular Biology of the Cell ( Garland Science 2008) 11
sterols Figure 10-5 Molecular Biology of the Cell ( Garland Science 2008) 12
Lipid composition varies with membrane Table 10-1 Molecular Biology of the Cell ( Garland Science 2008) 13
Asymmetry in the membranes Phospholipids with choline head groups enriched in extracytosolic leaflet (PC,SPH) Phospholipids with terminal amino group enriched in head group in cytosolic leaflet(pe, PS and also PI) Glycolipids in the outer leaflet Cholesterol in both Charge difference: majority of negative phospholipids (PS, PI) face inside Asymmetry is generated by flippases and counteracted by 'scramblases' 14
Lateral differentiation of membranes: phase separation Lipids in a mixtures in a single membrane may spontaneously segregate into different liquid phases Different fluidity Different dynamic properties Allows lateral partitioning of membrane components including proteins Figure 10-13 Molecular Biology of the Cell ( Garland Science 2008) 15
Lipid rafts 16
Introduction to biological membranes General functions and properties Membrane lipids Physical properties Distribution/asymmetry Membrane proteins transmembrane proteins Lipid-anchored proteins Peripheral proteins Distribution and signaling Phosphoinositides (Vesicular transport) Transporter 17
Membrane proteins Integral proteins Single pass trans-membrane protein; type I and II Polytopic (many passes through the membrane) Tail-anchored (ex. SNAREs) Loop-anchored (ex. Caveolin-1, reticulon homology domain) Lipid-anchored Peripheral proteins Lipid-associated proteins Protein-associated proteins 18
Transmembrane proteins α-helical TM domains can be detected on a hydrophobicity plot TM β-barrel protein (in bacterial and mitochondrial outer membranes) are more difficult to predict 19
Integral proteins that do not span the membrane include 20
Lipidated proteins (cytosolic face) Myristylation Fatty Acylation Prenylation Figure 10-20 Molecular Biology of the Cell ( Garland Science 2008) 21
Lipidated protein (cytosolic face) C14 fatty acid via amide bond to amino group of N-terminal glycin Added to cotranslationally to cytosolic non membrane proteins, a permanent modification. Sufficient for membrane binding only when combined with:1) Positive charge cluster,2) FA alkyl group, or 3) proteinprotein interactions Arf1 and c-src Exposed or hidden after reversible conformational change. Fatty acylation Typically C16 i.e. palmitic acid By thioester to cystein Reversible modification in cis-golgi, on soluble proteins and endodomains of transmembrane proteins Double FA-acyl group direct many proteins to lipid rafts Caveolin, influenza hemagglutinin. Prenylation Farnesyl (C15) or geranylgeranyl (C20) By thioether to cystein Added to cytosolic non membrane proteins. So called CaaX box usually at the Cterminus, a permanent modification Often combined with nearby FA acyl groups Two Ras isoforms: H ras farnesyl plus two FA acyl chains. K-ras Farnesyl plus cluster of positive charges Myristylation 22
Importance of protein lipidation They allow proteins to come on and off membranes and thus support dynamic processes during signal transduction, molecular sorting, membrane bending, vesicle formation, membrane recognition, etc. Functions and dynamics can be strictly regulated in time and place. Allow interaction of proteins with specific membranes only and with specific lipid micro-domains such as lipid rafts. 23
GPI-anchored proteins (extra-cytosolic) Chemical composition: PI phosphatidyl Several sugar inositol (GPI) anchor residues (N-acetyl glucose addition amine and mannose plus others) Phosphoethanolam ine connected by amide bond to Cterminus of protein. Glyco Topology: In the extracellular leaflet. The GPI anchor provides the only connection with the membrane. Distribution: Enriched in apical membranes of epithelial cells, Enriched in lipid rafts. Tail can be removed by phospholipase C, releasing the proteins. Examples: Thy-1 antigen Alkaline phosphatase Acetyl-choline esterase 24
Peripheral proteins Non-covalently attached to lipid head-groups or proteins in the membrane Complex mixture of proteins on both sides of a membrane Interactions are often transient and regulated Cytosolic side of PM is particularly rich in peripheral proteins: an extensive, dynamic cortex of actin, adaptor proteins, and other proteins (needed for membrane stability; local membrane specializations; connections with cytoskeleton; transmission of signals; trafficking of vesicles; cell shape and polarity determination; membrane curvature; endocytosis.) 25
Cytosolic face of the plasma membrane 26
Focal adhesion complex 27
Peripheral protein binding to the membrane Binding to other proteins Lipid-binding domain PIPs PH-domain FYVE motif C2-domain Curvature sensing domain BAR-domain ALPS-motif 28
Introduction to biological membranes General functions and properties Membrane lipids Physical properties Distribution/asymmetry Membrane proteins Integral proteins Lipid-anchored proteins Peripheral proteins Distribution and signaling Phosphoinositides (Vesicular transport) Transporter 29
Phosphatidylinositol phosphate(s) Inositol= cis-1,2,3,5-trans-4,6cyclohexanehexol Phosphoinositides (PIs) undergo phosphorylation/dephosphorylation through organelle-specific PI kinases and PI phosphatases in three positions of the inositol ring: 3,4 and 5. These combinations lead to different lipid products with distinct subcellular distributions. Phospholipases can cleave inositol-phosphate and release it in the cytosol as a second messenger 30
Phosphatidylinositol phosphate(s) In their specific locations, PIs control the correct timing and location of many important trafficking events. They do it via cytosolic proteins that bind to their inositol ring. These proteins include coat proteins, membrane fusion/fission proteins, membrane bending factors, enzymes, GTPase effectors, etc Most of these factors associate transiently with the membrane. 31
Phosphoinositides have preferred cellular locations PIP(4,5)P2 Plasma membrane PI(4)P Golgi complex PI(3)P Early endosomes PI(3,5)P2 Late endosomes They face the cytosolic compartment 32
PI-phosphorylation/ dephosphorylation pathways. s Mediated by a large family of PI-kinases and phosphatases s s From: Maria Antonietta De Matteis & Anna Godi Nature Cell Biology 6, 487 492 (2004) s 33
Phosphoinositide binding domains 34
Lipid transporters Can extract any kind of lipid from a membrane and deliver it to another membrane Bind lipid in hydrophobic pocket From: Nat Rev Mol Cell Biol. 2010 Oct;11(10):739-50 35
Osh4p structure Sterol-bound PI4P-bound 36
Example: generation of sterol gradient From: J Cell Biol(2011) 195, 965 78. 37