3.5.2010 Lecture<br /><ul><li>Transport from golgi to ER and ER to Golgi
How ? – Slide 25
Soluble protein can be recognized by transmembrane receptors
Transmembrane receptors have exit signals on cytosolic side of protein recognized by codimers
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  1. 1. 3.5.2010 Lecture<br /><ul><li>Transport from golgi to ER and ER to Golgi
  2. 2. How ? – Slide 25
  3. 3. Soluble protein can be recognized by transmembrane receptors
  4. 4. Transmembrane receptors have exit signals on cytosolic side of protein recognized by codimers
  5. 5. Codimers
  6. 6. Clathrin/adaptin
  7. 7. Sar1-GTP
  8. 8. Binds gtp
  9. 9. Begins recognition of exit signal on cargo receptor
  10. 10. Recruits COP proteins to site on membrane to form bud and make vesicle
  11. 11. Moves in anterograde to Golgi
  12. 12. Entire process begins with GTP bound to Sar,
  13. 13. when bound to GTP, it undergoes conformational change that stick out a hydrophobic part of protein that inserts itself into phospholipid bilayer
  14. 14. Sar1/GTP functional activats and recogizes COP II proteins
  15. 15. Signals coming back to ER
  16. 16. COP I codimers
  17. 17. because retrievel signal is recognized.
  18. 18. As proteins associate they tend to pinch off and make the pinchin a favorable reaction
  19. 19. Requires GTP
  20. 20. Codimers need to be lost so vesicles can be fused to target membrane
  21. 21. GAP proteins on Sar speed up hydrolysis of GTP to GDP
  22. 22. Quick summary
  23. 23. Gaps function in cytosol
  24. 24. Confo of Sar changes
  25. 25. Released from membrane
  26. 26. Causes dissociation of COP II which then bud off
  27. 27. What triggers exchange of Sar1?
  28. 28. GEF for Sar1 has been identified – Sec12
  29. 29. Returns Sar 1 to functional state
  30. 30. Cargo receptor
  31. 31. Clathrin and adaptin proteins
  32. 32. Adaptin acts as adapter, links cytosolic side of exit receptor to clathrin proteins
  33. 33. Clathrin is a triskelion molecule due to its structures, 3 large and 3 small subunits. Amino termini point inward, so when formed together, form hexagonal or pentagonal shape that forms a skeleton around a vesicles like a soccer ball. Stitching is clathrin and the coating is the PM
  34. 34. Energy is required to form this shape
  35. 35. When Clathrin binds adaptin starts to form bud
  36. 36. Dynamin recognizes clathrin bud and wraps itself around budding part of vesicle. With the use of GTP, will pinch membrane together and the vesicle is now released.
  37. 37. Seen in plasma membrane when
  38. 38. endocytosis takes place and goes to endomembrane system
  39. 39. Bud off golgi and go to endomembrane system
  40. 40. To uncoat vesicle
  41. 41. Remove adaptin from coated vesicle and release clathrin coated vesicle
  42. 42. Heatshock protein called hsp70 that is an ATPase, and it uses ATP hydrolysis to remove clathrin and adaptin from the exit receptors that they recognized.
  43. 43. Needs a helper called auxillin
  44. 44. Stimulates function of heatshock protein
  45. 45. Heatshock hydrolyzes ATP and realeases coat proteins from vesicle
  46. 46. Heatshock proteins are a large family of proteins found ubiquitously in cytosol and lumen of the ER/cytosol, but also important chaperone proteins as well as transport proteins.
  47. 47. Uncoated and now can fuse to a membrane
  48. 48. LDL – low density lipoproteins (slide 27)
  49. 49. We need to bring in cholesterol to cell . The LDL that contains cholesterol
  50. 50. Core of esterified molecules linked to fatty acids surrounded by phospholipid monolayer with cholesterol embedded. Small protein wraps around core as well as PL monolayer. Protein carries recognition sequence for LDL binding receptor.
  51. 51. Dalton protein that wraps around core and it carries a recognition sequence signal for LDL binding receptor
  52. 52. Cholesterol from blood, receptors recognize in membrane and bring in LDL via endocytosis
  53. 53. LDL binding site on LDL receptor recognizes LDL in EXCSpace
  54. 54. Adaptin protein AP-1 will recruit clathrin to localization of receptors.
  55. 55. Clathrin forms bud of membrane
  56. 56. Dynamin pinches off
  57. 57. Auxillin and heatshock uncoat and then will fuse with endosomes
  58. 58. In endosomes the ph is different enough (more acidic slightly) just enough to dissociate receptor from LDL. LDL is trafficked to lysosomes where hydrolases break it down and release cholesterol into cytosol to be reused by cell and receptor to be reused in the PM
  59. 59. This process of endocytosis happens all the time whether LDL Is present or not.
  60. 60. Cell recycles LDL receptors every 20min
  61. 61. Cell can regulate how many receptors are put back in membrane if cholesterol is high
  62. 62. Clathrin forms bud off membrane and dynamin pinches off, auxillin and heat shock work to uncoat, fuse with endosomes
  63. 63. In endo. Ph is different just enough to dissociate receptor from LDL, so the LDL is trafficked to lysosome where hydrolases break it down into cytosol and receptors are put back in membrane.
  64. 64. Mutations that occur in LDL receptors where they are made by cell but cannot bind to LDL in the blood so nothing is brought into the cell
  65. 65. Increases risk of heart attacks
  66. 66. Increase in Atherosclerosis
  67. 67. Retrograde transport – slide 28
  68. 68. In trans golgi, soluble protein from ER makes way to golgi, all the way through it, via COP II antereograde transport.
  69. 69. Within golgi, receptors recognize the KDEL sequence, the retrieval sequence for proteins to be maintained in the ER
  70. 70. ER retrieval/retention signal
  71. 71. KDEL – lysine, asparagine, glutamate, leucine
  72. 72. The 4 amino acids act as retrieval sequence and when folded properly, exposed and COP I recognizes EXC portion of the KDEL receptor, form a bud, uncoat from bud, targeted back to the ER
  73. 73. The Er binds to KDEL protein/same receptor lets go of receptor in ER is because of difference in pH.
  74. 74. ER has slightly less acidic ph than golgi
  75. 75. Golgi becomes less acidic causes association
  76. 76. As receptor is trafficked back to ER, neutral ph releases protein and now back in ER
  77. 77. Neutral pH released protein and it is now in the ER.
  78. 78. Another retention signal – arganine, any other amino acid, arginine sequence – used to keep proteins in ER as well and bring proteins back that have been out of ER.
  79. 79. Slide 29 – lysosomal proteins into clathrin-coated vesicles.
  80. 80. In golgi, after lysosomal proteins have undergone modifications, two signals are recognized by lysosomal targeting receptor
  81. 81. Manose-6-phosphate receptor
  82. 82. Recognition sites for M6P that has been placed to and modified in the ER on that lysosomal protein
  83. 83. On cytosolic side, there is a terminal end of receptor that links to an adapter protein
  84. 84. Adapter is recognized by clathrin
  85. 85. Clathrin forms bud to make vesicle
  86. 86. How is Manose-6-phosphate generated – slide 30
  87. 87. ER signal sequence that puts proteins into the ER as they are made
  88. 88. Once in ER and folded, N-glycosylation puts oligosaccharaides on that lysosomal protein
  89. 89. Once protein moves in golgi, in cis golgi, we see there is phosphorylation of this sugar that is place on N-terminus of asparagine,
  90. 90. Form a structure that is a sugar plus the phosphate
  91. 91. GlcNAc-P stands for N-acetylglucosamine phosphate
  92. 92. Recognition enzymes cleave off some of the sugar to leave the M6P present
  93. 93. As it moves through cis/medial/trans golgi, the phosphorylation acts as a recogntion sequence for enzymes that are gonna come in and cleave spme sugar away, exposing just M6P component of oligosaccharide tag and M6P is recognized as signal sequence that will be trafficked via the receptor to the lysosome.
  94. 94. Once fused with lysosome the M6P hydrolase is released into them due to high acidity ph5-5.5
  95. 95. Dissociation leads to removal of Pi from enzyme so it’s functional
  96. 96. The Pi just acts as a tag to get hydorlase to lysosome
  97. 97. M6P receptors are recycled back to golgi and used again for trafficking
  98. 98. This phosp. Event is the beginning of this transformation of protein to M6P
  99. 99. Slide 31 – how do vesicles go to where they need to go?
  100. 100. Motor proteins carry vesicles once budded form donor membrane to wherever they need to be
  101. 101. MP can recognize signal sequences on cargo itself, or recongize target signals within membrane of vesicle itself.
  102. 102. Phosphoinositides can be recognized by MPs in membrane and carried to vesicles
  103. 103. Centrosomes and mictrotubules extend out with + ends toward plasma membrane
  104. 104. Kinesin and dynein family MPs bind to vesicles as they bud and carry to certain places. Kinesin moves +, dynein moves –
  105. 105. Slide 32 – SNARES
  106. 106. Once vesicles form V-snares on donor membrane need to fuse with t-snares, accepting membrane, and these are specific proteins as well.
  107. 107. Specific v-snares to match with specific t-snare.
  108. 108. Slide 33 Snare Hypothesis
  109. 109. Start with bud form of vesicle from donor membrane with v-snare embedded
  110. 110. Coiled-coil protein bring vesicle to t-snares
  111. 111. Hyrolysis of nucleotide triphosphate
  112. 112. Rab associates with donor vesicle and in its active state is bound to GTP
  113. 113. Once vesicle is in it right places, vesicles need to be docked at membrane and must have recognition of proteins involved in fusion
  114. 114. Transport takes place.
  115. 115. Rab GTPase is involved with regulation of movement of vesicles to target membrane because if you remove it, the vesicles wont target and docking doesn’t occur
  116. 116. Rab hasn’t hydrolyzed GTP yet!
  117. 117. Remains bound to membrane in GTP bound state
  118. 118. 2 complexes that associate with vesicle that are on the target membrane and link with vesicle that help bring vesicles close to t snares so the v and t associate
  119. 119. Coiled coil tethering protein and teherting complex bring vesicle for snares to tether
  120. 120. Multipe subunit tethering protein
  121. 121. Dissociation
  122. 122. GTP hydrolysis takes place between association and fusion of vesicle
  123. 123. 2 family of proteins come in and dissociate v and t snares from each other
  124. 124. SNAPs and NSF proteins work to break apart associate between v and t SNARES
  125. 125. Slide 34 – How proteins are moved into mitochondria/specifically matrix
  126. 126. Begins in cytosol because the proteins to be trafficked into mitochondriaare synthesized by free ribosomes in cytosol
  127. 127. Once translated, immediately bound by heat shock proteins (Hsp70)
  128. 128. Heatshock protein 70 recognizes N-terminus alpha helix domain that is specific targeting to the mitochndria
  129. 129. TOM protein binds to recognition sequence, alpha helix, and links it to the pore in the mitochondira to allow for movement through the memrane (post translational transport)
  130. 130. Once protein moves to pore after recognized by receptor, TOM protein fuses with TIM protein (trans. Protein. Of inner mito membra) and makes a single large pore to bring protein to matrix of mitochondria.
  131. 131. Process requires energy because the heatshock proteins that keep the protein from folding in cytoplasm, requires the hydrolysis of ATP to be stripped off so protein can be moved through membrane
  132. 132. As moves through membran, signal peptidase cleaves signal and protein moves through
  133. 133. Hsp70 helps bind and pull protein through TIM and then the Hsp70 needs to be removed to be folded properly
  134. 134. Another HSP helps fold protein again using ATP and now we have a properly folded protein in matrix of mitochondria (Hsp60 folds to final conformation)
  135. 135. N-terminus alpha helix is positively charged amino acids that have hydrolxylated amino acids in between but can be recognized by this TOM receptor to begin post-translational import
  136. 136. Transport in and out of nucleus
  137. 137. Occurs through nuclear pores
  138. 138. Histones are made in cytoplasm but must move in some they can wind around chromosomes
  139. 139. Nuclear pores
  140. 140. Complex structure with core transporter protein that can be opened and closed
  141. 141. 2 ring subunits made of eight different proteins that form either side of the NP that links the nuclear envelope
  142. 142. called nucleoporins: strands of amino acids stick off of them and act as receptors to bind to proteins and entry into the nucleus
  143. 143. anchor proteins help hold nuclear pore in the membrane of nuclear membrane
  144. 144. Slide 37
  145. 145. Protein in cytoplasm with a NLS (a few basic amino acids clustered along anywhere along protein, when folded, exposed and recognized by proteins in cytosol that target the protein to nuclear pore
  146. 146. Importin protein recognizes NLS and carries protein to receptor sites in nuclearporins at nuclear pore and opens up nuclear pore to get into nucleus.
  147. 147. Pores are not open all the time
  148. 148. Once opened, protein and importin go into membrane
  149. 149. Ran – GTP binding site
  150. 150. In GTP bound form, strips importin protein from its cargo and allows cargo to be released into the nucleus
  151. 151. Ran binds imporitn and carries it out through nuclear pores
  152. 152. In cytosol, Ran is hydrolyzed by GAP proteins, so Ran is now bound to GDP
  153. 153. Dissociates from importin for reuse
  154. 154. GEFS in nucleus activate Ran to strip away importain and release protein in nucleus
  155. 155. GAPs in cytosol allow for importin to be reused for another type fo transport
  156. 156. Exportin acts with Ran to recognize nuclear export signal
  157. 157. NES bound by RAN GTP, carries out of nucleus into cytoplasm, hydrlized and releases protein and exportin can be recycled