Membrane targeting of proteins

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Chapter 3

• Membrane targeting of proteins
• By
• D. Thomas Rutkowski Vishwanath R. Lingappa

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3.1 Introduction

• Cells must localize proteins to specific
  organelles and membranes.
• Proteins are imported from the cytosol directly
  into several types of organelles.

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3.1 Introduction

• The endoplasmic reticulum (ER)
• is the entry point for proteins into the
  secretory pathway
• is highly specialized for that purpose
• Several other organelles and the plasma membrane
  receive their proteins by way of the secretory
  pathway.

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3.2 Proteins enter the secretory pathway by
translocation across the ER membrane (an overview)

• Signal sequences target nascent secretory and
  membrane proteins to the ER for translocation.
• Proteins cross the ER membrane through an aqueous
  channel that is gated.

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3.2 Proteins enter the secretory pathway by
translocation across the ER membrane

• Secretory proteins translocate completely across
  the ER membrane
• transmembrane proteins are integrated into the
  membrane.
• Before leaving the ER, proteins are modified and
  folded by enzymes and chaperones in the lumen.

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3.3 Proteins use signal sequences to target to
the ER for translocation

• A protein targets to the ER via a signal
  sequence, a short stretch of amino acids that is
  usually at its amino terminus.
• The only feature common to all signal sequences
  is a central, hydrophobic core that is usually
  sufficient to translocate any associated protein.

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3.4 Signal sequences are recognized by the signal
recognition particle (SRP)

• SRP binds to signal sequences.
• Binding of SRP to the signal sequence slows
  translation so that the nascent protein is
  delivered to the ER still largely unsynthesized
  and unfolded.

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3.4 Signal sequences are recognized by the signal
recognition particle (SRP)

• The structural flexibility of the M domain of
  SRP54 allows SRP to recognize diverse signal
  sequences.

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3.5 An interaction between SRP and its receptor
allows proteins to dock at the ER membrane

• Docking of SRP with its receptor brings the
  ribosome and nascent chain into proximity with
  the translocon.
• Docking requires the GTP binding and hydrolysis
  activities of SRP and its receptor.

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3.6 The translocon is an aqueous channel that
conducts proteins

• Proteins translocate through an aqueous channel
  composed of the Sec61 complex, located within the
  ER membrane.
• Numerous accessory proteins that are involved in
• Translocation
• Folding
• Modification associate with the channel

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3.7 Translation is coupled to translocation for
most eukaryotic secretory and transmembrane
proteins

• An interaction between the translocon and the
  signal sequence causes the channel to open and
  initiates translocation.
• The exact mechanism of translocation may vary
  from one protein to another.

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3.8 Some proteins target and translocate
posttranslationally

• Posttranslational translocation proceeds
  independently of both ribosomes and SRP.
• Posttranslational translocation is used
  extensively in yeast but is less common in higher
  eukaryotes.

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3.8 Some proteins target and translocate
posttranslationally

• The posttranslational translocon is distinct in
  composition from the cotranslational translocon,
  but they share the same channel.

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3.9 ATP hydrolysis drives translocation

• The energy for posttranslational translocation
  comes from ATP hydrolysis by the BiP protein
  within the ER lumen.
• The energy source for cotranslational
  translocation is less clear, but might be the
  same as for posttranslational translocation.

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3.9 ATP hydrolysis drives translocation

• Most translocation in bacteria occurs
  posttranslationally through a channel that is
  evolutionarily related to the Sec61 complex.

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3.10 Transmembrane proteins move out of the
translocation channel and into the lipid bilayer

• The synthesis of transmembrane proteins requires
  that transmembrane domains be
• recognized
• integrated into the lipid bilayer

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3.10 Transmembrane proteins move out of the
translocation channel and into the lipid bilayer

• Transmembrane domains exit the translocon by
  moving laterally through a protein-lipid
  interface.

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3.11 The orientation of transmembrane proteins is
determined as they are integrated into the
membrane

• Transmembrane domains must be oriented with
  respect to the membrane.
• The mechanism of transmembrane domain integration
  may vary considerably from one protein to another
• especially for proteins that span the membrane
  more than once

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3.12 Signal sequences are removed by signal
peptidase

• Nascent chains are often subjected to covalent
  modification in the ER lumen as they translocate.
• The signal peptidase complex cleaves signal
  sequences.

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3.13 The lipid GPI is added to some translocated
proteins

• GPI addition covalently tethers the C-termini of
  some proteins to the lipid bilayer.

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3.14 Sugars are added to many translocating
proteins

• Oligosaccharyltransferase catalyzes N-linked
  glycosylation on many proteins as they are
  translocated into the ER.

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3.15 Chaperones assist folding of newly
translocated proteins

• Molecular chaperones associate with proteins in
  the lumen and assist their folding.

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3.16 Protein disulfide isomerase ensures the
formation of the correct disulfide bonds as
proteins fold

• Protein disulfide isomerases catalyze disulfide
  bond formation and rearrangement in the ER.

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3.17 The calnexin/calreticulin chaperoning system
recognizes carbohydrate modifications

• Calnexin and calreticulin escort glycoproteins
  through repeated cycles of chaperoning.
• The cycles are controlled by addition and removal
  of glucose.

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3.18 The assembly of proteins into complexes is
monitored

• Subunits that have not yet assembled into
  complexes are retained in the ER by interaction
  with chaperones.

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3.19 Terminally misfolded proteins in the ER are
returned to the cytosol for degradation

• Translocated proteins can be exported to the
  cytosol.
• There they are
• ubiquitinated
• degraded by the proteasome
• a process known as ER-associated degradation.

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3.19 Terminally misfolded proteins in the ER are
returned to the cytosol for degradation

• Proteins are returned to the cytosol by the
  process of retrograde translocation.
• This is not as well understood as for
  translocation into the ER.

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3.20 Communication between the ER and nucleus
prevents the accumulation of unfolded proteins in
the lumen

• The unfolded protein response
• monitors folding conditions in the ER lumen
• initiates a signaling pathway that increases the
  expression of genes for ER chaperones
• The protein Ire1p mediates the unfolded protein
  response in yeast by becoming activated in
  response to conditions of cellular stress.

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3.20 Communication between the ER and nucleus
prevents the accumulation of unfolded proteins in
the lumen

• Activated Ire1p splices HAC1 mRNA.
• It results in the production of the Hac1 protein,
  a transcription factor that
• localizes to the nucleus
• binds to the promoters of genes with a UPR
  response element
• The unfolded protein response in higher
  eukaryotes has evolved more layers of control
  beyond those seen in yeast.

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3.21 The ER synthesizes the major cellular
phospholipids

• The major cellular phospholipids are synthesized
  predominantly on the cytosolic face of the ER
  membrane.

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3.21 The ER synthesizes the major cellular
phospholipids

• The localization of enzymes involved in lipid
  biosynthesis can be controlled by the cell to
  regulate the generation of new lipids.
• Cholesterol biosynthesis is regulated by
  proteolysis of a transcription factor integrated
  into the ER membrane.

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3.22 Lipids must be moved from the ER to the
membranes of other organelles

• Each organelle has a unique composition of
  lipids.
• This requires that lipid transport from the ER to
  each organelle be a specific process.
• The mechanisms of lipid transport between
  organelles are unclear.
• They might involve direct contact between the ER
  and other membranes in the cell.
• Transbilayer movement of lipids establishes
  asymmetry of membrane leaflets.

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3.23 The two leaflets of a membrane often differ
in lipid composition

• Movement of lipid molecules between the leaflets
  of a bilayer is required to establish asymmetry.
• Enzymes (flippases) are required for movement
  of lipids between leaflets.

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3.24 The ER is morphologically and functionally
subdivided

• The ER is morphologically subdivided into
  specialized compartments, including
• the rough ER for protein secretion
• the smooth ER for steroidogenesis and drug
  detoxification
• the sarcoplasmic reticulum for calcium storage
  and release

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3.24 The ER is morphologically and functionally
subdivided

• The functions of the smooth ER can be specialized
  according to the needs of the particular cell
  type.
• The ER may also be subdivided at the molecular
  level, in ways not morphologically evident.

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3.25 The ER is a dynamic organelle

• The extent and composition of the ER change in
  response to cellular need.
• The ER moves along the cytoskeleton.

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3.25 The ER is a dynamic organelle

• The mechanisms by which the ER expands and
  contracts and forms tubules have yet to be
  discovered.
• The signaling pathways that control ER
  composition are not yet understood but may
  overlap with the unfolded protein response.

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3.26 Signal sequences are also used to target
proteins to other organelles

• Signal sequences are used for targeting to and
  translocation across the membranes of other
  organelles.
• Mitochondria and chloroplasts are enclosed by a
  double membrane, with each bilayer containing its
  own type of translocon.
• Two distinct pathways target matrix proteins to
  peroxisomes.

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3.27 Import into mitochondria begins with signal
sequence recognition at the outer membrane

• Mitochondria have an inner and an outer membrane,
  each of which has a translocation complex.
• Import into mitochondria is posttranslational.

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3.27 Import into mitochondria begins with signal
sequence recognition at the outer membrane

• Mitochondrial signal sequences are recognized by
  a receptor at the outer membrane.

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3.28 Complexes in the inner and outer membranes
cooperate in mitochondrial protein import

• The TOM and TIM complexes associate physically,
  and the protein being imported passes directly
  from one to the other.
• Hsp70 in the mitochondrial matrix and the
  membrane potential across the inner membrane
  provide the energy for import.

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3.29 Proteins imported into chloroplasts must
also cross two membranes

• Import into chloroplasts occurs
  posttranslationally.
• The inner and outer membranes have separate
  translocation complexes that cooperate during the
  import of proteins.

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3.30 Proteins fold before they are imported into
peroxisomes

• Peroxisomal signal sequences are
• recognized in the cytosol
• targeted to a translocation channel
• Peroxisomal proteins are imported after they are
  folded.

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3.30 Proteins fold before they are imported into
peroxisomes

• The proteins that recognize peroxisomal signal
  sequences remain bound during import and cycle in
  and out of the organelle.
• Peroxisomal membranes originate by budding from
  the ER.