1. MCB: Exam 1 Review

2. Mueckler The Structure of Biological Membranes The Signal Hypothesis and the Targeting of Nascent Polypeptides to the Secretory Pathway Targeting of Nascent Polypeptides Outside of the Secretory Pathway

3. Functions of Cellular Membranes 1.Plasma membrane acts as a selectively permeable barrier to the environment Uptake of nutrients Waste disposal Maintains intracellular ionic milieu 2.Plasma membrane facilitates communication With the environment With other cells 3. Intracellular membranes allow compartmentalization and separation of different chemical reaction pathways Increased efficiency through proximity Prevent futile cycling through separation Protein secretion Mueckler1_MembraneStructure

4. Composition of Animal Cell Membranes Hydrated, proteinaceous lipid bilayers By weight: 20% water, 80% solids Solids: Lipid Protein Carbohydrate (~10%) Phospholipids responsible for basic membrane bilayer structure and physical properties Membranes are 2-dimensional fluids into which proteins are dissolved or embedded ~ 90% Mueckler1_MembraneStructure

5. Figure 10-2 Molecular Biology of the Cell (© Garland Science 2008) Structure of Phosphoglycerides All Membrane Lipids are Amphipathic Mueckler1_MembraneStructure

6. Figure 10-11a Molecular Biology of the Cell (© Garland Science 2008) (Hydrophobic) (Hydrophilic) Bilayers are Thermodynamically Stable Structures Formed from Amphathic Lipids Mueckler1_MembraneStructure

7. Lipid Bilayer Formation is Driven by the Hydrophobic Effect HE causes hydrophobic surfaces such as fatty acyl chains to aggregate in water Water molecules squeeze hydrophobic molecules into as compact a surface area as possible in order to the minimize the free energy state (G) of the system by maximizing the entropy (S) or degree of disorder of the water molecules  G =  –  S Mueckler1_MembraneStructure

9. Figure 1-12 Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008) Lipid Bilayer Formation is a Spontaneous Process Gently mix PL and water Vigorous mixing Mueckler1_MembraneStructure

10. Figure 10-8 Molecular Biology of the Cell (© Garland Science 2008) The Formation of Cell-Like Spherical Water-Filled Bilayers is Energetically Favorable Mueckler1_MembraneStructure

11. Phosphoglycerides are Classified by their Head Groups Phosphatidylethanolamine Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Ether Bond at C1 PS and PI bear a net negative charge at neutral pH Mueckler1_MembraneStructure

12. Figure 10-11b Molecular Biology of the Cell (© Garland Science 2008) PhosphoLipid Movements within Bilayers (µM/sec) (10 12 -10 13 /sec) (10 8 -10 9 /sec) Mueckler1_MembraneStructure

13. Pure Phospholipid Bilayers Undergo Phase Transition Below Lipid Phase Transition Temp Above Lipid Phase Transition Temp Gauche Isomer Formation All-Trans Mueckler1_MembraneStructure

14. Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008) A “Scramblase” Enzyme Catalyzes Symmetric Growth of Both Leaflets in the ER Mueckler1_MembraneStructure

15. Figure 10-16 Molecular Biology of the Cell (© Garland Science 2008) The Two Plasma Membrane Leaflets Possess Different Lipid Compositions Enriched in PC, SM, Glycolipids Enriched in PE, PS, PI Mueckler1_MembraneStructure

16. Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008) A “Flippase” Enzyme promotes Lipid Asymmetry in the Plasma Membrane Mueckler1_MembraneStructure

17. Figure 10-17a Molecular Biology of the Cell (© Garland Science 2008) Phospholipids are Involved in Signal Transduction PI-4,5P PI-3,4,5P 1. Activation of Lipid Kinases Mueckler1_MembraneStructure

18. 2. Phospholipases Produce Signaling Molecules via the Degradation of Phospholipids Mueckler1_MembraneStructure

19. Figure 10-17b Molecular Biology of the Cell (© Garland Science 2008) Phospholipase C Activation Produces Two Intracellular Signaling Molecules PI-4,5P I-1,4,5P Diacylglycerol Mueckler1_MembraneStructure

20. 3 Ways in which Lipids May be Transferred Between Different Intracellular Compartments Vesicle Fusion Direct Protein-Mediated Transfer Soluble Lipid Binding Proteins Mueckler1_MembraneStructure

21. Figure 10-19 Molecular Biology of the Cell (© Garland Science 2008) The 3 Basic Categories of Membrane Protein IntegralLipid- Anchored Peripheral (can also interact via PL headgroups) Single-PassMulti-pass Fatty acyl anchor GPI Anchor Transmembrane Helix Linker Domain  -Strands Mueckler1_MembraneStructure

22. Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008) Membrane Domains are “Inside-Out” Right-Side Out Soluble Protein Mueckler1_MembraneStructure

23. Figure 10-31 Molecular Biology of the Cell (© Garland Science 2008) Functional Characterization of Integral Membrane Proteins Requires Solubilization and Subsequent Reconstitution into a Lipid Bilayer Mueckler1_MembraneStructure

24. Transmembrane  -Helices 1.Right-handed 2.Stability in bilayer results from maximum hydrogen bonding of peptide backbone 3.Usually > 20 residues in length (rise of 1.5 Å/residue) 4.Exhibit various degrees of tilt with respect to the membrane and can bend due to helix breaking residues 5.Mostly hydrophobic side-chains in single-pass proteins 6.Multi-pass proteins can possess hydrophobic, polar, or amphipathic transmembrane helices H-Bond Mueckler1_MembraneStructure

25. Figure 10-22b Molecular Biology of the Cell (© Garland Science 2008) Transmembrane Domains Can Often be Accurately Identified by Hydrophobicity Analysis Mueckler1_MembraneStructure

26. Mueckler The Structure of Biological Membranes The Signal Hypothesis and the Targeting of Nascent Polypeptides to the Secretory Pathway Targeting of Nascent Polypeptides Outside of the Secretory Pathway

27. Signal-Mediated Targeting to the RER

28. Properties of Secretory Signal Sequences Hydrophobic Core N Mature Protein 8-12 Residues 15-30 Residues ++ Located at N-terminus 15-30 Residues in length Hydrophobic core of 8-12 residues Often basic residues at N-terminus (Arg, Lys) No sequence similarity cleavage

29. In Vitro Translation/Translocation System mRNA Rough microsomes Ribosomes tRNAs Soluble translation factors Low MW components Energy (ATP, creatine-P, creatine kinase) Reticulocyte or wheat germ lysate

30. Figure 12-37b Molecular Biology of the Cell (© Garland Science 2008) Isolation of Rough Microsomes by Density Gradient Centrifugation

31. In Vitro Translation/Translocation System mRNA + Translation Components + Amino acid* Protein* SDS PAGE

32. In Vitro Translation of Prolactin mRNA Prolactin is a polypeptide hormone (MW ~ 22 kd) secreted by anterior pituitary 1 2 3 4 5 6 7 8 MW (kd) 25 22 Lanes: 1.Purified prolactin 2.No RM 3.RM 4.No RM /digest with Protease 5.RM /digest with Protease 6.RM /detergent treat and add Protease 7.Prolactin mRNA minus SS + RM /digest with Protease 8.SS-globin mRNA + RM /digest with Protease 18 SDS Gel

33. Figure 12-44 Molecular Biology of the Cell (© Garland Science 2008) Post-Translational Translocation is Common in Yeast and Bacteria SecA ATPase functions like a piston pushing ~20 aa’s into the channel per cycle

34. Classification of Membrane Protein Topology Single-Pass, Bitopic Multipass, Polytopic

35. Figure 12-46 Molecular Biology of the Cell (© Garland Science 2008) Generation of a Type I Single-Pass Topology

36. Figure 12-47 Molecular Biology of the Cell (© Garland Science 2008) Generation of Type II and Type III Single Pass Topologies Type II Type III Post-translational Translocation

37. Figure 12-48 Molecular Biology of the Cell (© Garland Science 2008) Multipass Topologies are Generated by Multiple Internal Signal/Anchor Sequences Type IVa + – + – + – + –

38. Figure 12-49 Molecular Biology of the Cell (© Garland Science 2008) Multipass Topologies are Generated by Multiple Internal Signal/Anchor Sequences Type IVb + –

39. The Charge Difference Rule for Multispanning Membrane Proteins NH 2 COOH NH 2 COOH + + – + + NH 2 COOH NH 2 COOH + + + + cytoplasm – – – – – –

40. + Transmembrane Charge Inversion Disrupts Local Membrane Topology in Multipass Proteins NH 2 COOH + – – + 1 4 3 2 NH 2 COOH + – – + 1 4 3 2 1 2 3 4 NH 2 COOH 1 2 3 4 NH 2 COOH cytoplasm L1 L2 L3 L1 L2 L3 L1 L2 L3 – L1 L2 L3

41. Figure 12-51 Molecular Biology of the Cell (© Garland Science 2008) N-Linked Oligosaccharides are Added to Nascent Polypeptides in the Lumen of the RER

42. Disulfide Bridges are Formed in the RER by Protein Disulfide Isomerase (PDI)

43. Mueckler The Structure of Biological Membranes The Signal Hypothesis and the Targeting of Nascent Polypeptides to the Secretory Pathway Targeting of Nascent Polypeptides Outside of the Secretory Pathway

44. Figure 12-23 Molecular Biology of the Cell (© Garland Science 2008) Proteins are Incorporated Into Mitochondria Via Several Different Routes

45. Targeting to the Matrix Requires an N- Terminal Import Sequence

46. Figure 12-22 Molecular Biology of the Cell (© Garland Science 2008) N-terminal Import Sequences Form Amphipathic  Helices that Interact with the Tom20/22 Receptor Hydrophobic cleft

47. Protein Import into the Matrix Requires Passage Through Two Separate Membrane Translocons

48. Proteins Traverse the TOM and TIM Translocons in an Unfolded State

49. Targeting to the Inner Membrane Occurs Via 3 Distinct Routes Oxa1-MediatedStop-Transfer-MediatedTom70/Tim22/54-Mediated Multi-Pass Proteins Single-Pass Proteins Cytochrome oxidase subunit CoxVa ATP Synthase Subunit 9 ADP/ATP Antiporter

50. Cytochrome B2Cytochrome c Heme Lyase Targeting to the Intermembranous Space Occurs Via Two Distinct Pathways Direct Delivery Indirect IM Space Protease

51. Figure 12-27 Molecular Biology of the Cell (© Garland Science 2008) Targeting to the Outer Membrane Via the SAM Protein Complex ( S orting and A ssembly Machinery) (  -Barrell)

52. Figure 12-13 Molecular Biology of the Cell (© Garland Science 2008) Nuclear Import and Export Sequences are Recognized by Different Members of the Same Receptor Family (Keryopherins)

53. Figure 12-14 Molecular Biology of the Cell (© Garland Science 2008) Directionality is Conferred on Nuclear Transport by a Gradient of Ran-GDP/GTP Across the Nuclear Envelope

54. Figure 12-15 Molecular Biology of the Cell (© Garland Science 2008) Nuclear Import and Export Operate Via Reciprocal Use of the Ran-GDP/GTP Concentration Gradient