2 intracellular compartments

• Single-pass transmembrane protein

  A protein with a cleaved ER signal sequence and a single internal hydrophobic domain that remains in the lipid bilayer
• Internal ER signal sequence

  Can act as a start transfer sequence
• Single-pass transmembrane proteins

  • Can result when the protein has a single internal ER signal sequence which remains in the lipid bilayer
  • The protein's N-terminus can project into the cytosol (A) or into the ER lumen (B)
• Multipass transmembrane proteins

  Proteins that span the ER membrane multiple times
• Combinations of start-transfer and stop-transfer signals

  1. Determine the 'topology' of multipass transmembrane proteins

  2. Result in a protein that spans the ER membrane twice so that both N and C terminals project into the cytoplasm

  3. Multiple membrane spanning domains are possible with various final orientations of N and C terminals
• Rhodopsin

  A multipass membrane protein that is inserted into the ER during its translation
• Hydrophobic protein regions

  Act as alternating start and stop transfer signals so that rhodopsin can adopt its 7 membrane spanning orientation
• This would require 4 interactions with 4 different translocators working serially as the protein is translated
• 1. linked glycosylation

  Occurs on target asparagine (N) amino acids once a protein has been inserted into the ER lumen
• The 'N' in N-linked stands for asparagine, not the N-terminus of the protein, or a Nitrogen atom
• 1. linked oligosaccharides

  Are used as tags to mark the state of protein folding
• Calnexin

  1. An ER-membrane bound chaperone protein that binds to incompletely folded proteins with a single glucose remaining in their N-linked oligosaccharide chain

  2. The bound protein is folded via interaction with calnexin and other associated chaperones and then it is evaluated for proper folding before glucosidase trims the glucose to release the protein from calnexin

  3. If incompletely folded, a single glucose is added to the protein by glucosyl transferase and the cycle repeats
• Improperly folded proteins

  1. Are exported from the ER and degraded in the cytosol

  2. Interact with an array of molecules in the ER and are exported to the cytosol via a translocator

  3. E3 ubiquitin ligase adds ubiquitin monomers which form a polyubiquitin chain and target the protein for degredation by the proteasome
• Exocytosis

  Vesicles fuse with the plasma membrane and contents are released to the extracellular space
• Endocytosis

  A region of the plasma membrane pinches inwards capturing contents from the extracellular space for transport into the cell
• Transport vesicles

  • Move soluble proteins and membrane between compartments
  • Cargo molecules that resided within the inside space (lumen) of the donor compartment end up in the lumen of the target compartment
• Secretory pathway

  1. Movement of cargo molecules is termed 'secretion'

  2. The membrane-bound pathway through which vesicles move

  3. Works outwards (anterograde) and inwards (retrograde)
• Types of coated vesicle

  • Clathrin
  • COPI
  • COPII
• Clathrin

  Molecules form basketlike cages that help shape membranes into vesicles
• Clathrin triskelion

  Composed of 3 clathrin heavy chains and 3 light chains, triskelions merge to form the complex vesicle architecture
• Clathrin light chains

  Link to the actin cytoskeleton to provide the force for membrane budding and vesicle movement
• Adaptor proteins

  1. Select cargo into clathrin-coated vesicles

  2. Cargo receptors are plasma membrane spanning proteins that bind cargo molecules and interact with adaptor proteins which recruit clathrin to begin vesicle formation
• Adaptor Protein 2 (AP2)

  A complex of 4 subunits that binds to the phosphoinosotide PI(4,5)P2 (a membrane lipid) and to different cargo receptor proteins in the plasma membrane
• Each AP2 complex binds 4x PI(4,5)P2 lipids (although only one is illustrated)
• Binding of the open AP2 complex

  Begins the induction of membrane curvature that will result in clathrin-coated vesicle formation
• Phosphoinositides (PIPs)

  Lipids which mark organelles and membrane domains
• Phosphatidylinositol (PI)

  Is modified by addition of phosphates at the 3 and 4 position carbon -OH groups to produce the phosphoinositide PI(3,4)P2
• PI and PIP kinases and phosphatases

  Carry out the lipid modifications to produce different PIP configurations
• Different adaptor proteins recognise the different PIP configurations and are recruited to membrane regions and organelles where those particular PIPs accumulate
• Different types of PIPs are associated with different membranes within the secretory pathway
• These PIPs are interconvertible because the different PIP kinase and PIP phosphatase enzymes reside in specific membrane regions
• Dynamin

  1. A cytoplasmic protein which induces tight curvature in the neck region of budding clathrin-coated vesicles and aids in vesicle budding

  2. Multiple dynamin molecules assemble into a spiral around the neck of a forming bud and pinch the neck so that the non-cytoplasmic leaflets of the membrane flow together
• Formation of a COPII-coated vesicle for cargo transport from ER to Golgi

  1. Active SAR1-GTP binds to the ER membrane and recruits the COPII adaptor coat protein SEC23 which, in turn, recruits SEC24

  2. SEC24 then binds to any of a number of different ER membrane-bound cargo receptors

  3. Inactive SAR1-GDP binds to the SAR1-guanine exchange factor (SAR1-GEF) in the ER membrane and GDP is switched for GTP which activates SAR1

  4. SEC13 and SEC31 then form the outer layer of the COPII coat

  5. Continued recruitment of SAR1 and SEC proteins eventually results in budding of the COPII-coated vesicle
• Rab proteins

  • Guide transport vesicles to their target membrane
  • Think of Rab proteins as address labels
  • Rab proteins are GTPases that circulate between the cytoplasm and a membrane
• Rab cycle

  There are Rab GAPs and Rab GEFs that drive the 'Rab cycle'
• Tethering of a transport vesicle to a target membrane

  1. Rab effector proteins in target membranes interact with active Rab-GTP in vesicle membranes to initiate docking

  2. SNARE proteins then interact to bring vesicles into contact with target membranes

  3. Rab-GTP is then hydrolysed to Rab-GDP and becomes solubilized by binding to RabGDP dissociation inhibitor (GDI) for reuse
• SNAREs

  • Proteins that mediate membrane fusion
  • Vesicle SNARE (v-SNARE) and Target-membrane SNARE (t-SNARE)
  • Tight binding of v-SNAREs and t-SNAREs forces water out and creates a water-free environment for membrane fusion
• Rab5 membrane domain

  Is constructed on an endosome to recruit clathrin-coated vesicles
• Rab cascades

  1. Can change the identity of an organelle

  2. RabA is initially activated by RabA-GEF (green GEF) and recruits its Rab effectors (blue) to a target membrane

  3. One of the effectors might be RabB-GEF (blue GEF) which activates RabB and, in turn RabB recruits its Rab effectors (green)

  4. One of the RabB effectors might be a GTPase activating protein, e.g. RabA-GAP which causes hydrolysis of RabA-GTP to RabA-GDP so that it is removed from the membrane

  5. In this manner, membranes can change identity, e.g. between early-, and late-endosome