How does a protein get to the correct cellular location?

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How does a protein get to the correct cellular
location?

• Membrane and organelle proteins contain targeting
  (sorting) signals in their amino acid sequence.
• Targeting signals are recognized during or after
  the protein is translated - special machinery
  recognizes the signal and translocates the
  protein to its correct location

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Examples of protein targeting signals
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Proteins are targeted to different compartments
in different ways
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Proteins that are targeted to the nucleus,
mitochondria, chloroplasts and peroxisomes are
synthesized on free ribosomes as soluble
polypeptides
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Proteins that are targeted to the cell surface,
Golgi and Lysosomes are synthesized on ER
membrane bound ribosomes and move through the
secretory pathway
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Overview of secretory pathway
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All proteins encoded by nuclear DNA are first
translated on free cytoplasmic ribosomes

• Soluble proteins and proteins targeted to the
  mitochondria, chloroplasts and peroxisomes are
  completely synthesized on free ribosomes
• Translation of Integral membrane proteins,
  secreted proteins, and proteins in the ER, Golgi,
  and lysosomes are synthesized on ribosomes bound
  to the ER membrane
• The subunits on free and ER bound ribosomes are
  identical

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What happens when protein targeting doesnt work?

• I-cell disease caused by defect in lysosomal
  targeting
• Many hydrolytic enzymes fail to be targeted to
  lysosomes and are secreted from cells
• Psychomotor retardation, skeletal abnormalities
• Average lifespan 8 years

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Zellwenger syndrome

• Peroxisomal targeting defect
• Peroxisomal enzymes accumulate in cytosol
• Neural, cardiovascular, renal, adrenal
  dystrophies
• Accumulate very long chain fatty acids
• Cataracts, glaucoma, retinal detachment
• Average lifespan - 12 weeks

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Protein TargetingHow do secreted proteins get
to the ER membrane?
Gunther BlobelNobel Prize 1999"for the
discovery that proteins have intrinsic signals
that govern their transport and localization in
the cell"
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Early experiments An N-terminal signal sequence
on nascent secretory proteins targets synthesis
to the ER and is then cleaved.
Translation of secretory mRNAs in cell free
protein synthesis system produces full length
proteins with intact signal sequence
Adding microsomes (ER membranes) to system causes
ribosomes to bind to membranes, translocation of
the protein to the lumen and cleavage of signal
sequence
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Signal sequences usually contain 1 or more
charged amino acids followed by a stretch of
hydrophobic residues
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The ER targeting mechanism requires two special
receptor proteins
What gets the ribosomes with secretory protein
mRNA's to bind to the ER membranes?
1. Signal recognition particle (SRP) 2. SRP
receptor
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Translation of secretory mRNA begins on free
ribosomes

• N-terminal signal sequence emerges from ribosome
  tunnel
• Signal recognition particle (SRP) binds to the
  emerging signal sequence from the ribosome

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• SRP is a ribonucleoprotein
• 300 base RNA molecule
• 6 proteins

• Methionine "whiskers" on P54 subunit bind to the
  hydrophobic signal sequence on the emerging
  polypeptide

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Cryo-EM map SRP and 80S ribosomeNature 427, 808
- 814 (26 February 2004
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SRP ribosome interaction
EFS elongation factor binding site
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Exam study recommendation

• Some questions on exam are to test the
  understanding of functions.
• e.g. What would be the effect of a loss of
  function mutation in the signal binding part of
  the Signal Recognition particle?

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SRP receptor initiates the interaction of signal
sequences with the ER membrane

• Receptor is an a,b dimer b subunit is an
  intrinsic membrane protein
• a-subunit initiates binding of ribosome SRP to
  ER membrane

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SRP/SRP receptor dissociates from signal sequence

• Ribosome binds to translocon
• Signal sequence binds to translocon. Translocon
  gate opens
• Signal sequence inserts into translocon central
  cavity w/ N-terminus toward cytosol

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• Polypeptide chain elongates signal sequence
  cleaved and degraded in ER lumen
• Peptide chain elongation extrudes protein into ER
  lumen

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• Sec63 complex promotes Hsc70 chaperone (BiP)
  binding to growing chain

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Ribosome dissociates and is released from
membrane when protein is completed
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What controls the insertion of nascent secretory
proteins into the translocon?

• The P54 subunit of the Signal Recognition
  Particle is a GTPase
• So is the a-subunit of the SRP receptor

GTP binding to both proteins produces
conformational changes required for tight
docking to the membrane
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GTP hydrolysis initiates protein transport into
the ER
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GTP hydrolysis powers 1) dissociation of SRP, SRP
receptor from translocon, 2) opening of
translocon gate, 3) transfer of signal sequence
to translocon
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SRP and SR stimulate each other's GTPase
activity. GTP hydrolysis triggers
unidirectional targeting of ribosome/cargo
binding to the Sec61a translocation pore.
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What other GTP hydrolysis mechanisms power
protein translocation into the ER?
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Peptide bond formation
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Secretory proteins move from the Rough ER lumen
through Golgi complex and then to cell surface by
vesicle mediated transport
This is driven by energy released during protein
translation
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How do intrinsic membrane proteins get inserted
into the ER membrane?
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Topologies of some integral membrane proteins
synthesized on the rough ER
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Most cytosolic transmembrane proteins have an
N-terminal signal sequence and an internal
topogenic sequence
Type I protein
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A single internal signal-anchor sequence directs
insertion of single-pass Type II transmembrane
proteins
Type II protein, no N-terminal signal sequence
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Multipass transmembrane proteins have multiple
topogenic sequences
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After insertion into the ER membrane, some
proteins are transferred to a GPI anchor
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Post-translational modifications and quality
control in the rough ER

• Newly synthesized polypeptides in the membrane
  and lumen of the ER undergo five principal
  modifications
• Formation of disulfide bonds
• Proper folding
• Addition and processing of carbohydrates
• Specific proteolytic cleavages
• Assembly into multimeric proteins

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Disulfide bonds are formed and rearranged in the
ER lumen
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Most proteins synthesized in the Rough ER are
glycosylated by a core oligosaccharide that is
linked to asparagine residues(N-linked
glycosylation)
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The core oligosaccharide used for N-linked
glycosylation is assembled onto the
polyisoprenoid lipid, dolichol pyrophosphate
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Dolichol is an poly - isoprenoid compound
synthesized by the same metabolic route as
cholesterol.

• In vertebrate tissues, dolichol contains 18-20
  isoprenoid units (90-100 carbons total).

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Formation of the Core Oligosaccharide on Dolichol
Phosphate starts in the cytosol and is completed
in the ER lumen
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N-linked glycosylation occurs during protein
translocation via the membrane bound protein
oligosaccharide transferase
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Core Glycosylation and Trimming in the ER lumen
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Oligosaccharide Transferase
Glucose and Mannose Trimming
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Correct folding of newly made proteins is
facilitated by several ER proteins that bind to
oligosaccharides

• Calnexin and Calreticulin are Ca binding
  proteins that bind to glucosylated
  oligosaccharides of incompletely folded proteins
• promote association with protein disulfide
  isomerase which facilitates formation of correct
  disulfide bonds
• prevent incompletely folded proteins from
  irreversible aggregation before disufide bond
  formation and intitial folding occurs

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Bip, Calnexin and Calreticulin binding promote
folding of adjacent areas while correct disulfide
bonds are formed
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How do small molecules move through cell
membranes?
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Ions and most small molecules move across
membranes through intrinsic membrane proteins
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Fundamental Rule of Membrane Transport
1.Movement of molecules across a membrane with
the concentration gradient (from high to low
concentration) does not require metabolic energy
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Fundamental Rule of Membrane Transport 2.
Movement of molecules across a membrane against
its concentration gradient requires a source of
metabolic energy
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There are two main sources of energy used to
transport molecules across cell membranes
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There are two main sources of energy used to
transport molecules across cell membranes

• 1. Energy derived from ATP hydrolysis
• 2. Energy derived from the electrochemical
  potential (which is many times produced by ATP
  hydrolysis)

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Cells and organelles maintain a wide variety of
electrochemical gradients across their membranes
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There are 3 primary types of transport proteins
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ATP powered pumps are ATPases - energy of ATP
hydrolysis moves ions or small molecules across a
membrane against a concentration gradient and/or
an electrical potential (Active Transport)
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Channel proteins move ions and hydrophilic
molecules down their concentration or electrical
gradient they are highly regulated and very
fast (facilitated diffusion)
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Transporters move molecules across membranes
using existing concentration gradients (glucose,
sucrose amino acids)
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Uniporters transport a single molecule down its
concentration gradient
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Symporters cotransport specific molecules against
its concentration gradient along with another
molecule down its electrochemical gradient
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Antiporters cotransport a specific molecule
against its concentration gradient along with
another molecule down its electrochemical
gradient from the other side of the membrane
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Glucose moves into most cells from the blood
stream through uniporters

• Transport of glucose is highly specific
• Rate of facilitated diffusion is much higher than
  passive transport through the bilayer
• Each cell type has a type of glucose transporter
  that is adapted to its particular function

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Glucose transporters change their conformation on
binding their substrate
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If the concentration gradient of glucose shifts
from outside to inside, the transporter will work
in reverse
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There are four major classes of ATP driven pumps
they are responsible for generating the
electrochemical potential of cell membranes
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P-class pumps are phosphorylated as part of the
transport cycle
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V-class and F-class ATPases pump protons
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F-class pumps are primarily ATP synthetases
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ABC transporters move lipophilic molecules and
function as flippases