Chapter 15 Intracellular compartments and transport

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Chapter 15Intracellular compartments and
transport

• Membrane enclosed organells
• Protein sorting
• Vesicular transport
• Secretory pathways
• Endocytic pathways

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• Each eucaryotic cell is subdivided into many
  enclosed compartments where many metabolic
  processes are carried out
• Separating cellular compartments is necessary for
  the reactions not to mix and be carried out
  simultaneously
• Many enzymes, reactions, substrates, and products
  are actively synthesized and degraded

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• The most prominent organelle is the nucleus
  surrounded by a double membrane (nuclear
  envelope) which communicates with the cytosol via
  nuclear pores
• The outer nuclear membrane forms endoplasmic
  reticulum (ER) with ribosomes attached
• Smooth ER is used for special function proteins
  for example synthesis of steroid hormones
  (adrenal glands)
• Golgi apparatus-receives and modifies proteins
  from ER
• Lysosomes-contain digestive enzymes for
  degradation
• Mitochondria-oxydative phosphorylation
• Fig 15-2

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• How did the organelles evolved?
• Nuclear membrane and ER are thought to evolve
  through invagination of the plasma membrane
• Mitochondria are thought to originate by
  engulfing bacterium by a primitive eucaryotic
  cell
• Fig 15-3 -4

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• Protein sorting
• Proteins are synthesized in cytosol
• They have to be delivered to different cellular
  compartments
• Presence of a sorting signal in each of the
  proteins directs them to a particular compartment
  (lack of such a signal allows the protein to stay
  in the cytosol)
• Sorting
• Nuclear pores serve as selective gates
• Transport across the membranes (into ER, mt)
• Transport by vesicles by pinching off
• Fig 15-5

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• Signal sequences direct proteins to the correct
  destination compartment
• 15-60 amino acids long
• Usually on the N-terminal end of the protein
• Signal sequence is removed once the protein
  transported
• The function of signal sequences was determined
  experimentally
• Fig 15-6

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• Nuclear pores
• Proteins enter nucleus by nuclear pores
• Nuclear pores-two way traffic proteins-IN
  mRNA-OUT
• Nuclear pore itself is a large structure composed
  of approximately 100 proteins
• Small molecules can pass freely
• RNA, proteins, and other large molecules require
  sorting signal
• Fig 15-8

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• The signal directing proteins to the nucleus is a
  nuclear localization signal (NLS)
• The nuclear transport receptor (NTR) is located
  in the cytosol
• NTR binds to NLS of the protein and it helps to
  direct the protein to the nuclear pore using
  fibrils
• Protein is transported actively using GTP
  hydrolysis
• NTR dissociates and returns to cytosol for re-use
• Proteins transported to the nucleus remain in
  their fully folded conformation (distinct
  feature)
• Fig 15-9

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• Import proteins to the mitochondria
• Some proteins are synthesized in mt
• Some proteins have a signal sequence directing
  them to mt
• Proteins needing to be transported inside the
  mitochondria are translocated through the outer
  and inner membrane of mitochondria
• Chaperones inside mitochondria help to pull and
  fold the transported proteins properly
• Fig 15-10

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• Unlike the proteins that enter nucleus or mt,
  proteins that enter ER are not completely
  synthesized at the time of translocation, thus
  the presence of ribosomes on ER
• A common pool of ribosomes is used to synthesize
  both, proteins to stay in cytoplasm and these
  destined to ER
• ER destined proteins posses ER signal sequence
• Once the ER protein synthesis is completed,
  released ribosomes join the common pool of
  ribosomes
• Fig 15-12

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• ER signal sequence is guided by two components
• Signal recognition particle (SRP)
• SRP receptor in the ER membrane
• Protein is guided to the ER membrane by the SRP
• SRP binds to its receptor in the ER membrane
• SRP dissociates
• Protein is threaded and synthesized through the
  ER lumen via translocation channel
• Fig 15-13

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• The ER signal sequence is degraded by signal
  peptidase during translocation
• The peptide is released to the lumen of ER
• The proteins is folded in its correct
  conformation once the C-terminus passed the
  translocation channel
• Fig 15-14

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• Some proteins will stay in ER as transmembrane
  proteins
• If only one segment is the membrane spanning, the
  translocation starts using the N-terminal signal
  sequence but the process is stopped by the
  stop-transfer sequence
• The signal sequence is clipped off
• As a result, the transmembrane protein has a
  defined orientation with the N-terminus in the ER
  lumen and the C-terminus in the cytosol
• Fig 15-15

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• ER is the first step to transport proteins into
  the Golgi apparatus
• In Golgi, continuous budding and fusion of the
  vesicles occurs
• This process allows for the protein modification
• Glycosylation (addition of sugar)
• Disulfide bond formation for protein
  stabilization
• Fig 15-17

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• Vesicle budding is driven by assembly of protein
  coat its function
• Shape of the vesicle
• Helps to capture molecules
• Steps in formation of the vesicle
• Cargo receptor binds protein
• Adaptin bind receptors
• Clathrin coat is formed
• Dynamin pinches off the vesicle
• Fig 15-19

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• Released vesicles must reach their destination
  and fuse with the proper membrane
• Precise transport requires molecular markers
  identifying the vesicle and target membrane
• Family of proteins SNARES
• v-SNARE on the vesicle
• t-SNARE on the target membrane
• It is thought that there are many different
  complementary pairs of t-SNARES and v-SNARES,
  each specific to one kind of transport
• Fig 15-20

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• After docking, v-SNARE and t-SNARE wrap around
  each other tightly and help to pull both
  membranes together
• Membrane fusion is initiated with help of other
  proteins
• Fig 15-21

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• Secretory pathways
• Newly made proteins are transported from ER via
  Golgi to the cell surface
• This process of fussing membranes and releasing
  proteins is called exocytosis
• Most proteins are modified in ER
• Formation of disulfide bonds by oxidation
• proteins becomes more stable
• resistant to pH changes or enzymes
• Glycosylation is a covalent attachment of
  oligosaccharides function
• Protects from degradation
• Holding in ER till the protein properly folded
• Transport signal for packaging into appropriate
  vesicle

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• Glycosylation
• Sugars are added as branched oligosaccharides
• N-linked sugars are most common-sugar transferred
  to the amino group of the amino acid aspargine
• Oligosaccharides undergo further processing in
  Golgi
• Fig 15-22

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• Proteins destined to stay in ER have an ER
  retention signal
• Other proteins are transported to Golgi
• Further transport of proteins depends on their
  proper folding
• Chaperones help proteins to fold properly
• Antibody molecules composed of 4 polypeptide
  chains are folded in ER
• Chloride channel
• Protein misfolding (caused by mutation)
• Protein accumulates in ER
• Cystic fibrosis, degradation of lungs
• Fig 15-23

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• Golgi apparatus
• A collection of flattened, membrane enclosed sacs
• Each Golgi has
• the CIS entry face (faces ER)
• the TRANS exit face (faces cellular membrane)
• Proteins travel from ER to Golgi enclosed in
  vesicles
• Protein destination-cell surface or another
  cellular compartment
• Fig 15-24

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• Studying location and movement of proteins in the
  cell
• Green fluorescent protein (GFP) can be fused to a
  protein of interest (viral coat protein)
• A high temp the fusion protein labels the ER
• B decrease of temp protein accumulates at the ER
  exit site
• C protein moves to Golgi
• D vesicles expelled to plasma membrane
• Fig 15-27

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• Secretory proteins are released from Golgi by
  exocytosis
• Constitutive pathway (default)
• Operates continuously
• Non-selective pathway
• Regulated pathway
• Only in cells specialized for secretion
• Produce large quantities of proteins hormones,
  enzymes
• Proteins are stored and accumulate near plasma
  membrane
• Extracellular signal is needed for the transport
  vesicle to fuse with the membrane and release its
  content
• Glucose level increased-signal to pancreatic
  cells to release insulin
• Fig 15-28

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• Conditions in Golgi are acidic and increased
  calcium ions allows proteins to aggregate and
  pack tightly
• Protein concentration is up to 200 fold
• Large amounts of protein can be released quickly
  when the appropriate signal is received

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• Receptor mediated endocytosis-molecules bind to
  their receptors on the cell surface and enter the
  cell as clathrin coated vesicle
• Cholesterol transported in blood as a LDL
  (low-density-lipoprotein) complex
• LDL binds to its receptor
• Ingested by the receptor-mediated endocytosis
• Acidic conditions in the endosome (the receptor
  is released and returned to the cell membrane)
• LDL is delivered to the lysosome
• Cholesterol is released by enzymes
• Free cholesterol is ready for membrane formation
• If the LDL receptor is mutated and
  non-functional, cholesterol accumulates in blood
  and clogs arteries
• Fig 15-32

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• Endosome
• Sorting station for endocytosed molecules
• Very acidic
• Receptors release their cargo here
• Destination of a receptor in the endosome
• Recycling-returned to the membrane
• Degradation-travel to lysosome and digested
• Transcytosis-transport bound cargo from one
  extracellular space to another
• Fig 15-33

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• Lysosomes-sites for intracellular digestion
• The surrounding membrane keeps the enzymes in and
  separates the pH (cell pH 7.2)
• 40 enzymes are active in acidic environment
  (pH 5)
• ATP driven H pump allows to keep low pH in the
  lysosome (high concentration of H inside the
  lysosome)
• Degrade proteins, nucleic acids,
  oligosaccharides, phospholipids
• Amino acids, sugars, nucleotides are transported
  out of the lysosome to the cytosol
• Fig 15-34