Intracellular Compartments

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Intracellular Compartments

• ER, Golgi Complex, Endosomes, Lysosomes,
  Peroxisomes

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Interconnections of Organelles

• Many metabolic activities occur within organelles
• A collection of membranous structures involved in
  transport within the cell. The main components of
  the endomembrane system are endoplasmic
  reticulum, Golgi bodies, vesicles, cell membrane
  and nuclear envelope. Members of the endomembrane
  system pass materials through each other or
  through the use of vesicles.

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Endomembrane Continuity Transport
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Check this out

• http//academic.brooklyn.cuny.edu/biology/bio4fv/p
  age/endomem.htm

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Biosynthesis in ER

• 50 90 of cells membrane is ER
• Structural, functional, secreted proteins
  synthesized in ER
• RER
• Lipids (triacylglycerols, cholesterol, others)
  synthesized in ER
• SER

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Rough ER

• Ribosomes attached to cytosolic side
• Transitional elements (TEs) form transition
  vesicles that shuttle proteins, lipids to Golgi
  complex
• Proteins enter endomembrane system
• Inserted into lumen cotranslationally
• Or imported into the ER posttranslationally
• (when destined for peroxisomes, mitochondria,
  chloroplasts)

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Microsomes

• Microsomes form from fractionation of SER RER
• In vitro artifact used to study enzymes
• Liver cells a common source
• Homogenization
• Differential centrifugation isolates microsomes
• Nuclei, mitochondria settle out at 10,000g
• Microsomes at 100,000g

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Microsomes
http//homepages.gac.edu/cellab/chpts/chpt7/figur
e7-3.html
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Intracellular Sorting of Proteins
Destined for endomembrane system or for export
from cell.
Destined for cytoplasm or other organelles.
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How Does the Cell Determine Which Pathway to Use?

• Gunter Blobel (Nobel Prize, 1999) David
  Sabatini signal hypothesis
• Intrinsic molecular signal identifies which
  proteins are to go to each location
• To ER ER signal sequence on N-terminus of
  polypeptide directs ribosome-mRNA-polypeptide
  complex to anchor at protein dock on ER surface

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Smooth ERFunctions

• Drug detoxification
• Hydroxylation of hydrophobic substances makes
  them soluble in H20, more easily excreted from
  the body
• Cytochrome P-450 enzymes of SER in liver
  (inducible)
• Elimination of barbiturates (experimental
  evidence)
• Aryl hydrocarbon hydroxylase (Cyt P-450)
• Metabolizes steroids, drugs, toxins including
  phenobarbital
• Often creates products often more toxic than
  substrate
• Converts potentially carcinogenic cmpds into
  active forms
• Cigarette smoke is potent inducer of enzyme
• Benzo(a)pyrene (precarcinogen) induces enzyme

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Smooth ERFunction cont.

• Carbohydrate metabolism
• Breaks down glycogen in liver cells
• Glucose-6-phosphatase bound to SER
• Glu-6-phosphate H20 ? Glu Pi
• Glu released from cell
• Calcium storage
• Muscle cell sarcoplasmic reticulum
• Ca released for muscle contraction ATPases
  pump Ca back into lumen for muscle relaxation.

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The Role of the Smooth ER in theCatabolis of
Liver Glycogen
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Calcium Regulation in Cells
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SER Function cont.

• Biosynthesis of phospholipids
• Most occurs in ER, distributed to organelles
• Enzymes located on cytosol side of SER lipids
  are inserted in that monolayer
• Vesicles made from ER reflect lipid content
• Flippases (phospholipid translocators) move some
  lipids to inner layer
• Phospholipid exchange proteins move lipids from
  ER to mitochondria, cholorplasts, plasma membrane

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The Golgi Complex

• Flattened membrane-bounded cisternae, disk-shaped
  sacs
• Part of endomembrane system
• Transport vesicles
• Bud off membranes, move to other areas
• Transport proteins, lipids to other organelles
• Coated vesicles (covered with proteins that
  promote curvature of membrane for vesicle
  formation.) Coat removed before fusion with
  membranes.
• Clathrin, COPI, COPII

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Golgi Structure
(cis-Golgi network)
(coated)
(coated)
trans-Golgi network)
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Immunochemical Staining of a Golgi Complex
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Polarity of Golgi Complex

• Different functions in CGN, TGN and medial
  cisternae
• Distinct enzymes present in each area
• Coated vesicles differ
• Vesicles that bud from ER for transport of
  proteins, lipids to CGN coated with COPII
• Vesicles from CGN or medial cisternae coated with
  COPI
• Vesicles from TGN coated with COPI and clathrin

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Protein Glycosylation

• Much of protein processing carried out within ER
  and Golgi complex involves glycosylation
• Addition of carbohydrate side chains to specific
  aa forming glycoproteins
• Core glycosylation is first stepER
• N-linked glycosylation
• O-linked glycosylation
• Synthesized on lipid, transferred to protein
• Subsequent enzyme-catalyzed reactions modify
  oligosaccharide side chain

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Steps of Glycosylation and Subsequent
Modification of Proteins
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N-linked Glycosylation Assembly of Core
Oligosaccharide and Transfer to Protein
Part 1
(N-acetylglucosamine)
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N-linked Glycosylation Assembly of Core
Oligosaccharide and Transfer to Protein
Part 2
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Additional Modification of Protein

• Second stage of N-glycosylation
• Calnexin, Calreticulin proteins in ER
• UGGT acts as sensor for proper folding
• Further modifications in cis face, medial
  cisternae
• Terminal glycosylation in Golgi
• Glycosylation occurs on lumenal surface of
  membranes.
• Binding with plasma membrane maintains location
  of carbs on outer surface

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Protein Trafficking

• The problem synthesized proteins must be
  directed to the proper locations in organelles
  and plasma membrane
• Solution Each protein contains specific tag
  that targets the location
• Tag can be specific aa sequence, an
  oligosaccharide side chain, a hydrophobic domain
• Some lipids are tagged and put into vesicles

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Trafficking through the Endomembrane System
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How to Retrieve Proteins to Remain in ER or Golgi
Complex

• Most likely AA tags retain proteins in both
  organelles
• Formation of large complexes exclude them from
  transport vesicles
• In Golgi length of membrane-spanning domains
  determines how far it moves through organelle.
  Membrane is thicker in trans than in cis region.

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Targeting of Soluble Lysosomal Proteins to
Endosomes Lysosomes

• In Golgi soluble proteins destined to lysosome,
  the mannose residues are phosphorylated forming
  oligosaccharide containing mannose-6-phosphate.
  Tag
• TGN Mannose-6-phosphate receptors are bound to
  membrane. Bind to oligosaccharides destined to
  endosomes which become/bind to lysosomes.

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Targeting of Soluble Lysosomal Enzyme to
Endosomes and Lysosomes by a Mannose-6-Phosphate
Tag
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Secretory Pathways Transport Molecules to
Exterior of Cell

• Transport from ER ? Golgi ? secretory vesicles
  and secretory granules
• Constitutive secretion
• Continuous discharge of vesicles at plasma
  membrane
• Regulated secretion
• Controlled rapid release of substance in response
  to extracellular signal

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Secretory Pathways

• Constitutive secretion
• Mucus, glycoproteins of plasma membrane
• Exocytosis
• Regulated secretion
• Vesicles remain close to plasma membrane
• Zymogen granules
• Signal leads to exocytosis
• Neurotransmitters, insulin, digestive enzymes of
  acinar cells of pancreas

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Zymogen Granules