Advanced Cell Biology
2014 1nd Semester
Department of Animal Science
Chungbuk National University
5th Lecture
1st week : Introduction
3rd week :Research Strategies For Cell Biology
5nd week : Nucleus, Transcription and Splicing
7nd ...
• Eukaryotic cells have an elaborate system of
internal membrane-bounded structures
called organelles.
• Each organelle:
–...
• An organelle comprises one or more
membrane-bounded compartments.
• Organelles may act autonomously or in
cooperation to...
Needs of Transport System..
• The vesicles form by budding from an
organelle’s surface.
• They subsequently fuse with the target
membrane of the accep...
Exocytic pathway
• All eukaryotes have the same complement of
core exocytic compartments:
– the endoplasmic reticulum
– th...
Endocytic pathway
• Extracellular material can be taken into cells by several
different mechanisms.
• The low pH and degra...
Endocytic and biosynthetic-secretory pathways
Transport vesicles
(Ten or more chemically distinct, membrane-enclosed compa...
The biosynthetic-secretory and endocytic pathways
March 30, 2006 Pabio552, Lecture 2 13
A 2. ER translocation is co-translational
March 30, 2006 Pabio552, Lecture 2 14
March 30, 2006 Pabio552, Lecture 2 15
Please review the signal hypothesis and mechanisms of co-translational translocation...
Vesicles
• Transport vesicles move proteins and other
macromolecules from one membrane-bounded
compartment to the next alo...
• Proteins destined for transport to one compartment are
sorted away from:
– resident proteins
– proteins that are destine...
Various types of coated vesicles
Golgi apparatus
Plasma
ER and
Golgi Cisternae
COPII-coated vesicles :
ER to Golgi
• ER membrane proteins activate Sar1p
GTPase.
• Activate Sar1p bring togerther a
trans...
COPI-coated vesicles :
Golgi to the ER
• COPI coat assembly is triggered by a
membrane-bound GTPase called ARF.
• ARF recr...
Clathrin-coated vesicles :
Golgi to endosome or
endocytosis
• COPI coat assembly is triggered by a
membrane-bound GTPase c...
Assembly of a clathrin coat
triskelion
Coated pits
and vesicles
on the cytosolic
surface of membranes
Freeze-etch
36 trisk...
Adaptin binds to cargo receptor and clathrin triskelion
Dynamin pinches of the vesicles
GTPase
Shibire mutant
has coated pits
but no budding off
of synaptic vesicles
ARF proteins: COPI&clathrin
Sar1 protein: COPII
GTP causes Sar1 to
Bind to membrane
Assembly and disassembly of coat by GT...
Vesicle delivery : Rab GTPases as molecular ‘Zip’ Code
- Transport vesicle anchor one of 60
varieties of Rab
- Vesicle-ass...
SNARE proteins guide vesicular transport
20 SNAREs, v-SNAREs, t-SNAREs
SNAREs specify compartment identity and control specificity
4 a helices
in trans-SNARE
complexes
Rab proteins ensure the specificity of vesicle docking
>30 Rabs
On cytosolic surface
C-terminal regions are variable:
Bind...
SNAREs may mediate membrane fusion
SNARE complex
After docking
The entry of enveloped viruses into cells
HIV
Similar to SNAREs
Proteins leave the ER in COPII-coated transport vesicles
ER exit sites
(no ribosomes)
Selective
process
Only properly folded and assembled proteins can leave the ER
Chaperones cover up exit signals
Homotypic membrane fusion
to form vesicular tubular clusters
The Golgi apparatus synthesizes sphingolipids, establishing a gradient of
sphingolipids and cholesterol and bilayer thickn...
An enzyme transfers the branched “core oligosaccharide” rich in mannose from dolichol
to the side chain of asparagine (sin...
Golgi apparatus glycosidases remove some sugars and
glycosyltransferases add sugars to remodel oligosaccharide side chains.
ER retrieval signals: KKXX in ER membrane proteins,
KDEL sequence in soluble ER resident proteins
Membrane proteins in Gol...
Targeting Protein and Membrane from TGN
The trans-Golgi network or TGN is a cluster of membrane-bounded tubules and vesicl...
Central sorting and distribution point for membranes and cargo
coming through the secretory pathway and destined for lysos...
Lysosome and Endosome
Endosomes : sorting compartments between the plasma membrane and lysosomes
Lysosomes contain a varie...
Endocytosis
- Cells utilize many different mechanism for endocytosis
- In phagocytosis and clathrin-mediated endocytosis, ...
Phagocytosis
Ingestion of large particles such as
bacteria, foreign bodies, and remnants of
dead cells
Four step in Phagoc...
Phosphatidylinositol (PO) is important regulator of Endocytosis
Macropinocytosis
- Many Cell ingest extercellular fluid in large endocytic structure called macropinosomes
Caveolae mediated Endocytosis
Caveole : enriched in cholestrol
Stabillized by the protein called caveolin
Histochemical stains: biochemical
Compartmentalization of the Golgi
Functional
compartmentalization
Transport through the Golgi may occur by vesicular
transport or cisternal maturation (not mutually exclusive)
Collagen rod...
Summary
1. Vesicular transport, biosynthetic-secretory and
endocytic pathways;
2. Coated vesicles;
3. Coat assembly and di...
Cell biology Lecture 5
Cell biology Lecture 5
Cell biology Lecture 5
Cell biology Lecture 5
Cell biology Lecture 5
Cell biology Lecture 5
Cell biology Lecture 5
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Cell biology Lecture 5

  1. 1. Advanced Cell Biology 2014 1nd Semester Department of Animal Science Chungbuk National University 5th Lecture
  2. 2. 1st week : Introduction 3rd week :Research Strategies For Cell Biology 5nd week : Nucleus, Transcription and Splicing 7nd week : Membrane and Channel 9nd week : Membrane Trafficking 11nd week : Cell Signaling 13nd week : Cytoskeleton 15nd week : Cell Cycle
  3. 3. • Eukaryotic cells have an elaborate system of internal membrane-bounded structures called organelles. • Each organelle: – has a unique composition of (glyco)proteins and (glyco)lipids – carries out a particular set of functions Why cell need Membrane Trafficking?
  4. 4. • An organelle comprises one or more membrane-bounded compartments. • Organelles may act autonomously or in cooperation to accomplish a given function. • In the endocytic and exocytic pathways, cargo proteins are transferred between compartments by transport vesicles.
  5. 5. Needs of Transport System..
  6. 6. • The vesicles form by budding from an organelle’s surface. • They subsequently fuse with the target membrane of the acceptor compartment. Vesicles
  7. 7. Exocytic pathway • All eukaryotes have the same complement of core exocytic compartments: – the endoplasmic reticulum – the compartments of the Golgi apparatus – post-Golgi transport vesicles
  8. 8. Endocytic pathway • Extracellular material can be taken into cells by several different mechanisms. • The low pH and degradative enzymes in endosomes and lysosomes are important in processing some endocytosed material.
  9. 9. Endocytic and biosynthetic-secretory pathways Transport vesicles (Ten or more chemically distinct, membrane-enclosed compartments)
  10. 10. The biosynthetic-secretory and endocytic pathways
  11. 11. March 30, 2006 Pabio552, Lecture 2 13 A 2. ER translocation is co-translational
  12. 12. March 30, 2006 Pabio552, Lecture 2 14
  13. 13. March 30, 2006 Pabio552, Lecture 2 15 Please review the signal hypothesis and mechanisms of co-translational translocation in the Alberts’ textbook!
  14. 14. Vesicles • Transport vesicles move proteins and other macromolecules from one membrane-bounded compartment to the next along the exocytic and endocytic pathways. • Coats formed from cytoplasmic protein complexes help to: – generate transport vesicles – select proteins that need to be transported
  15. 15. • Proteins destined for transport to one compartment are sorted away from: – resident proteins – proteins that are destined for other compartments • Transport vesicles use tethers and SNAREs to dock and fuse specifically with the next compartment on the pathway. • Retrograde (backward) movement of transport vesicles carrying recycled or salvaged proteins compensates for anterograde (forward) movement of vesicles.
  16. 16. Various types of coated vesicles Golgi apparatus Plasma ER and Golgi Cisternae
  17. 17. COPII-coated vesicles : ER to Golgi • ER membrane proteins activate Sar1p GTPase. • Activate Sar1p bring togerther a transmembrane cargo receptor • COPII coats deforms the membrane into a bud • After budding, coat component promotes hydrolysis of GTP bound to Sar1p
  18. 18. COPI-coated vesicles : Golgi to the ER • COPI coat assembly is triggered by a membrane-bound GTPase called ARF. • ARF recruits coatomer complexes, and disassembly follows GTP hydrolysis. • COPI coats bind directly or indirectly to cargo proteins that are returned to the endoplasmic reticulum from the Golgi apparatus.
  19. 19. Clathrin-coated vesicles : Golgi to endosome or endocytosis • COPI coat assembly is triggered by a membrane-bound GTPase called ARF. • ARF recruits coatomer complexes, and disassembly follows GTP hydrolysis. • COPI coats bind directly or indirectly to cargo proteins that are returned to the endoplasmic reticulum from the Golgi apparatus.
  20. 20. Assembly of a clathrin coat triskelion Coated pits and vesicles on the cytosolic surface of membranes Freeze-etch 36 triskelions 12 pentagons 6 hexagons Inner layer binds adaptins
  21. 21. Adaptin binds to cargo receptor and clathrin triskelion
  22. 22. Dynamin pinches of the vesicles GTPase Shibire mutant has coated pits but no budding off of synaptic vesicles
  23. 23. ARF proteins: COPI&clathrin Sar1 protein: COPII GTP causes Sar1 to Bind to membrane Assembly and disassembly of coat by GTPases Coat-recruitment GTPases GTPase works like a timer And cause disassembly shortly After the budding is completed
  24. 24. Vesicle delivery : Rab GTPases as molecular ‘Zip’ Code - Transport vesicle anchor one of 60 varieties of Rab - Vesicle-associated GEF activate Rab - Rab-GTP recruits tethering and fusion proteins - After Membrane fusion, GAP activates and hydrolysis Rab-GTP - Binds to GDI and return to donor membrane
  25. 25. SNARE proteins guide vesicular transport 20 SNAREs, v-SNAREs, t-SNAREs
  26. 26. SNAREs specify compartment identity and control specificity 4 a helices in trans-SNARE complexes
  27. 27. Rab proteins ensure the specificity of vesicle docking >30 Rabs On cytosolic surface C-terminal regions are variable: Bind to other proteins, including GEFs
  28. 28. SNAREs may mediate membrane fusion SNARE complex After docking
  29. 29. The entry of enveloped viruses into cells HIV Similar to SNAREs
  30. 30. Proteins leave the ER in COPII-coated transport vesicles ER exit sites (no ribosomes) Selective process
  31. 31. Only properly folded and assembled proteins can leave the ER Chaperones cover up exit signals
  32. 32. Homotypic membrane fusion to form vesicular tubular clusters
  33. 33. The Golgi apparatus synthesizes sphingolipids, establishing a gradient of sphingolipids and cholesterol and bilayer thickness from low in the ER to high in the plasma membrane. Transport through the Golgi apparatus: Membrane lipids, integral membrane proteins, and soluble proteins in the lumen move from the cis-Golgi through the stacks to the trans-Golgi.
  34. 34. An enzyme transfers the branched “core oligosaccharide” rich in mannose from dolichol to the side chain of asparagine (single letter abbreviation N) as the protein enters the lumen of the ER. An amide bond couples the first sugar to the side chain
  35. 35. Golgi apparatus glycosidases remove some sugars and glycosyltransferases add sugars to remodel oligosaccharide side chains.
  36. 36. ER retrieval signals: KKXX in ER membrane proteins, KDEL sequence in soluble ER resident proteins Membrane proteins in Golgi and ER have shorter TM domains (15 aa) Cholesterol pH controls affinity of KDEL receptors
  37. 37. Targeting Protein and Membrane from TGN The trans-Golgi network or TGN is a cluster of membrane-bounded tubules and vesicles adjacent to the trans-most stack of the Golgi apparatus.
  38. 38. Central sorting and distribution point for membranes and cargo coming through the secretory pathway and destined for lysosomes, endosomes, and plasma membrane. Roles of Trans-Golgi Networks
  39. 39. Lysosome and Endosome Endosomes : sorting compartments between the plasma membrane and lysosomes Lysosomes contain a variety of hydrolytic enzymes that degrade proteins, lipids, polysaccharides, and nucleic acids taken into the cell by endocytosis (see next section) as well as many cellular molecules that turn over normally.
  40. 40. Endocytosis - Cells utilize many different mechanism for endocytosis - In phagocytosis and clathrin-mediated endocytosis, cell surface receptor selectively bind macromolegules to be internalized
  41. 41. Phagocytosis Ingestion of large particles such as bacteria, foreign bodies, and remnants of dead cells Four step in Phagocytosis 1. Attachments 2. Engulfment - Formation of phagocuytic cup - Growth of actin filaments 1. Fusion With Lysosome 2. Degradation
  42. 42. Phosphatidylinositol (PO) is important regulator of Endocytosis
  43. 43. Macropinocytosis - Many Cell ingest extercellular fluid in large endocytic structure called macropinosomes
  44. 44. Caveolae mediated Endocytosis Caveole : enriched in cholestrol Stabillized by the protein called caveolin
  45. 45. Histochemical stains: biochemical Compartmentalization of the Golgi Functional compartmentalization
  46. 46. Transport through the Golgi may occur by vesicular transport or cisternal maturation (not mutually exclusive) Collagen rods Scales in algae
  47. 47. Summary 1. Vesicular transport, biosynthetic-secretory and endocytic pathways; 2. Coated vesicles; 3. Coat assembly and disassembly, budding, dynamin, coat-recruitment GTPases; 4. Targeting and fusion by Rab GTPases, SNAREs; 5. ER to Golgi: COPII, folding, fusion (cluster), retrograde; 6. Golgi apparatus structure and polarity; 7. Continuation of glycosylation; 8. Compartmentalization of Golgi cisternae; 9. By now we have introduced gated transport, transmembrane transport and vesicular transport.

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