Endomembrane system

• 3 general transport methods in endomembrane system

  1. Transport materials from donor to recipient compartment
  2. Transport materials out of the cell (secretory pathway)
  3. Transport materials into the cell (endocytic pathway)
• Transportation of materials from donor to recipient compartment

  • membrane-bound vesicles shuttle materials between organelles or from one part of the organelle to another
  • Vesicles bud from the donor
  • Transport in a directional manner using motor proteins and the cytoskeleton
  • vesicles fuse with the membrane of the recipient compartment
  • cargo is released, vesicle membrane fuses into recip. membrane
  • escaped resident proteins from the donor can be returned
• From donor to recipient

  • Proteins (membrane, secreted, enzymes) can be directed to their destination with sorting signals recognized by receptors in the membrane of budding vesicles:
  • amino acid sequences of the protein being transported
  • attached oligosaccharides
• Secretory pathway (biosynthetic pathway)

  Biomolecules are synthesized in the ER and modified by the Golgi complex before being constitutively or regulatorily secreted.

  • constitutive secretion
  • most cells use constitutive secretion
  • materials are CONTINUALLY transported in secretory vesicles until they reach the plasma membrane where the vesicle fuses with the membrane
  • regulated secretion
  • materials are stored in membrane-bound compartments and are only released in response to a particular stimuli
• Endocytic pathway

  Materials move from the outer surface of the cell to compartments within the cell (endosomes and lysosomes)

  • endosome:
  • materials are taken up and transported to early endosomes for sorting
  • Late endosomes are more acidic than early ones
  • Late endosomes fuse with lysosomes to deliver cargo meant for degradation
  • Lysosome:
  • Hydrolytic (digestive) enzymes and acidic pH
  • Roles in breakdown of material and organelle turnover

  
• Autoradiography

  Follows the location of radioactively labelled materials in a cell.

  Pulse-chase experiment: examines a process that takes place over time eg. determines the path of enzymes throughout their synthesis and secretion
• Pulse-chase experiment steps for following the path of an enzyme

  Step 1: Pulse

  • radio-labelled amino acids are incorporated in the digestive enzymes being synthesized. Cells are only exposed to radio-labelled amino acids for a short time (pulse)

  Step 2: Chase

  • Transfer cells to media with only un-labelled amino acids. During this time, the cell will synthesize un-labelled enzymes.

  Radio-labelled enzymes are tracked from synthesis to secretion

  
• Approaches to studying endomembranes - mutant phenotypes

  • Characterize phenotypes resulting from mutant or deleted gene (s)
  • Provides insight into the normal function of the protein

  ex.

  A) vesicle formation to Golgi

  
• Endoplasmic reticullum (ER)


  a system of membranes and vesicles that encloses the lumen (separated from the cytosol)

  • ER divided into smooth and rough ER
• ER

  A) lumen

  
• Rough ER

  • has ribosomes bound to the cytosolic membrane surface
  • composed of network of cisternae (flattened sacs)
  • Continuous with the outer membrane of the nuclear envelope
• Smooth ER

  • lacks ribosomes
  • composed of interconnected, curved, tubular membranes
  • Continuous with the rough ER
• ER structure again

  type out smooth/rough ER

  A) rough ER

  B) smooth ER

  C) nucleus

  
• Smooth ER functions

  • synthesis of steroid hormones (derived from cholesterol)
  • synthesis of membrane lipids
  • Detoxification of organic compounds of the liver
  • sequestering calcium ion in skeletal and cardiac muscle (role in muscle contraction)
• Rough ER functions

  • role in protein secretion
  • protein synthesis
  • initiating addition of sugars to proteins
• Sites of protein synthesis: RER ribosomes

  • 1/3 proteins made on rough ER
  • Proteins made through co-translation translocation: peptides move into the lumen of the ER as it is still being synthesized by the ribosome

  Makes:

  • secreted proteins
  • integral membrane proteins and soluble proteins that reside in the compartments of the endomembrane system
• co-translation translocation: peptides move into the lumen of the ER as it is still being synthesized by the ribosome
• Sites of protein synthesis: free ribosomes

  • 2/3 of proteins
  • proteins are released into the cytosol and remain there or...
  • peripheral proteins of the cytosolic surface of membranes
  • proteins transported to the nucleus, mitochondria, chloroplast, and peroxisomes
• RER ribosomes and free ribosomes are structurally and functionally the same
• synthesis of secreted/soluble proteins through co-translational translocation

  1.

  • All protein synthesis begins on a free ribosome.
  • signal sequence of protein at the N-terminal composed of 6-15 hydrophobic amino acids that helps direct the ribosome to the membrane
  • SRP (signal recognition particle) recognizes and binds to signal sequence. SRP binding will halt protein synthesis
• synthesis of secreted/soluble proteins through co-translational translocation


  2.

  • SRP directs this complex to the ER membrane using interactions with the SRP receptor
  • Ribosome and its polypeptide are transferred from SRP to translocon (protein pore with plug in ER membrane)
  • translocon's plug is displaced when in contact with hydrophobic signal sequence
• synthesis of secreted/soluble proteins through co-translational translocation


  3.

  • translocation through the pore; polypeptide enters the ER lumen
  • Upon termination, ribosome is released
  • Signal sequence is removed by signal peptidase
  • protein chaperones (BiP) aid in protein folding
• synthesis of integral membrane proteins through co-translational translocation



  1.

  • SRP recognizes and binds to the hydrophobic transmembrane domain as the signal sequence and brings it to the ER membrane using interactions with the SRP receptor
  • Transmembrane domains do not pass through the pore, but instead directly enter the lipid bilayer
• synthesis of integral membrane proteins through co-translational translocation



  2.

  • As polypeptides pass through the translocon, a gate in the pore opens and allows proteins to segregate themselves according to their solubility properties.
  • either sorted to the aqueous pore or into the hydrophobic lipid bilayer
• Translocon gate for integral proteins:

  
• Glycosylation in the rough ER

  carbohydrate groups attached to proteins have roles as binding sites, aid in proper folding and stabilization, and sorting/directing proteins to different cellular compartments

  • N-linked glycosylation: linkage to asparagine
  • initiated in the RER
  • O-linked glycosylation: linkage to serine or threonine
  • initiated in golgi
• N-linked glycosylation in the rough ER

  • First seven sugars are transferred lipid carrier (dolichol pyrophosphate) embedded into the ER membrane. Assembly occurs on the cytosolic side and sugars are added by glycosyltransferase
  • lipid carrier and oligosaccharide is flipped and the remaining sugars are assembled onto a lipid carrier on the cytosolic side before it is flipped back into the lumen to be added
  • Completed oligosaccharide is transferred to an asparagine of a polypeptide with the sequence Asn-n-Ser/Thr.
  • transferred by oligosaccharyltransferase
• N-linked glycosylation in the rough ER

  • First seven sugars are transferred lipid carrier (dolichol pyrophosphate) embedded into the ER membrane. Assembly occurs on the cytosolic side and sugars are added by glycosyltransferase
  • lipid carrier and oligosaccharide is flipped and the remaining sugars are assembled onto a lipid carrier on the cytosolic side before it is flipped back into the lumen to be added
  • Completed oligosaccharide is transferred to an asparagine of a polypeptide with the sequence Asn-n-Ser/Thr.
  • transferred by oligosaccharyltransferase
• quality control for misfolded proteins

  1. glucosidase I and II remove two glucoses
  2. glycoprotein with one glucose is recognized by calnexin
  3. removal of another glucose
  4. Incompletely folded proteins are recognized by UGGT which adds a glucose back so that calnexin recognizes it and folding may try again
  5. properly folded proteins exist
  6. improperly folded proteins are degraded by lysosome
• Exiting the ER

  1. membrane vesicles with cargo bud from ER
  2. transport vesicles fuse with each other to form larger vesicles called vesicular tubular carriers (VTC) in the ERGIC region (intermediate golgi)
  3. Vesicles bud and move through the cis and trans golgi
• Trans golgi network

  sorting station where proteins are segregated into different types of vesicles
• cis golgi network


  sorting station that distinguishes between proteins that need to be shipped back to the ER and those that should proceed to the TGN
• Protein modification w/ golgi

  Proteins leaving ER are sequentially modified

  • order that sugars incorporated depends on the location of the glycotransferases in the membrane of the golgi
  • o-linked glycosylation occurs in golgi
• Vesicular transport model

  golgi cisternae are stable and vesicles carrying cargo bud from one compartment and fuse with the next

  • evidence:
  • golgi cisternae have different enzymes
  • lost of vesicles bud from the edges of the golgi cisternae
• cisternal maturation model

  cisternae form from vesicles budding from the ER that accumulate to generate the cis face and move towards the trans face, maturing as they move

  • Evidence:
  • drugs blocking vesicle formation at ER leads to disappearance of Golgi complex
  • certain molecules move from cis or trans without appearing in vesicles