Protein%20trafficking%20between%20membranes

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Chapter 4

• Protein trafficking between membranes
• By
• Graham Warren Ira Mellman

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4.1 Introduction

• Eukaryotic cells have an elaborate system of
  internal membrane-bounded structures called
  organelles.
• Each organelle
• has a unique composition of (glyco)proteins and
  (glyco)lipids
• carries out a particular set of functions

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4.1 Introduction

• An organelle comprises one or more
  membrane-bounded compartments.
• Organelles may act autonomously or in cooperation
  to accomplish a given function.
• In the endocytic and exocytic pathways, cargo
  proteins are transferred between compartments by
  transport vesicles.

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4.1 Introduction

• The vesicles form by budding from an organelles
  surface.
• They subsequently fuse with the target membrane
  of the acceptor compartment.

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4.1 Introduction

• Transport vesicles can selectively
• include material destined for transfer
• exclude material that must remain in the
  organelle from which they bud
• Selective inclusion into transport vesicles is
  ensured by signals in a proteins amino acid
  sequence or carbohydrate structures.
• Transport vesicles contain proteins that target
  them specifically to their intended destinations
  with which they dock and fuse.

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4.2 Overview of the exocytic pathway

• All eukaryotes have the same complement of core
  exocytic compartments
• the endoplasmic reticulum
• the compartments of the Golgi apparatus
• post-Golgi transport vesicles

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4.2 Overview of the exocytic pathway

• The amount and organization of exocytic
  organelles varies from organism to organism and
  cell type to cell type.
• Each organelle in the exocytic pathway has a
  specialized function.
• The endoplasmic reticulum is the site for the
  synthesis and proper folding of proteins.

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4.2 Overview of the exocytic pathway

• In the Golgi apparatus, proteins are
• Modified
• Sorted
• carried by the post-Golgi transport vesicles to
  the correct destination.
• Cargo transport to the plasma membrane occurs
• directly by a constitutive process or
• indirectly by a regulated process.
• This involves temporary storage in secretory
  granules until the cell receives an appropriate
  stimulus.

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4.3 Overview of the endocytic pathway

• Extracellular material can be taken into cells by
  several different mechanisms.
• The low pH and degradative enzymes in endosomes
  and lysosomes are important in processing some
  endocytosed material.

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4.4 Concepts in vesicle-mediated protein transport

• Transport vesicles move proteins and other
  macromolecules from one membrane-bounded
  compartment to the next along the exocytic and
  endocytic pathways.
• Coats formed from cytoplasmic protein complexes
  help to
• generate transport vesicles
• select proteins that need to be transported

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4.4 Concepts in vesicle-mediated protein transport

• Proteins destined for transport to one
  compartment are sorted away from
• resident proteins
• proteins that are destined for other compartments
• Transport vesicles use tethers and SNAREs to dock
  and fuse specifically with the next compartment
  on the pathway.
• Retrograde (backward) movement of transport
  vesicles carrying recycled or salvaged proteins
  compensates for anterograde (forward) movement of
  vesicles.

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4.5 The concepts of signal-mediated and bulk flow
protein transport

• Soluble secretory proteins, especially those
  secreted in large amounts, may not require
  specific signals to traverse the exocytic pathway.

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4.5 The concepts of signal-mediated and bulk flow
protein transport

• Sorting signals may be restricted to membrane
  proteins and endocytosed receptors
• particularly those that are targeted to some
  intracellular destinations, such as lysosomes.
• Some soluble proteins have signals that allow
  them to interact with receptors that mediate
  their transport to lysosomes.

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4.6 COPII-coated vesicles mediate transport from
the ER to the Golgi apparatus

• COPII vesicles are the only known class of
  transport vesicles originating from the
  endoplasmic reticulum.
• Assembly of the COPII coat proteins at export
  sites in the endoplasmic reticulum requires a
  GTPase and structural proteins.

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4.6 COPII-coated vesicles mediate transport from
the ER to the Golgi apparatus

• Export signals for membrane proteins in the
  endoplasmic reticulum are usually in the
  cytoplasmic tail.
• After scission, COPII vesicles may cluster, fuse,
  and then move along microtubule tracks to the
  cis-side of the Golgi apparatus.

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4.7 Resident proteins that escape from the ER are
retrieved

• Abundant, soluble proteins of the endoplasmic
  reticulum (ER) contain sequences (such as KDEL or
  a related sequence).
• These sequences allow them to be retrieved from
  later compartments by the KDEL receptor.

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4.7 Resident proteins that escape from the ER are
retrieved

• Resident membrane proteins and cycling proteins
  are retrieved to the ER by a dibasic signal in
  the cytoplasmic tail.
• The ER retrieval signal for type I transmembrane
  proteins is a dilysine signal.
• Type II transmembrane proteins have a diarginine
  signal.

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4.8 COPI-coated vesicles mediate retrograde
transport from the Golgi apparatus to the ER

• COPI coat assembly is triggered by a
  membrane-bound GTPase called ARF.

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4.8 COPI-coated vesicles mediate retrograde
transport from the Golgi apparatus to the ER

• ARF recruits coatomer complexes, and disassembly
  follows GTP hydrolysis.
• COPI coats bind directly or indirectly to cargo
  proteins that are returned to the endoplasmic
  reticulum from the Golgi apparatus.

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4.9 There are two popular models for forward
transport through the Golgi apparatus

• Transport of large protein structures through the
  Golgi apparatus occurs by cisternal maturation.
• Individual proteins and small protein structures
  are transported through the Golgi apparatus
  either by cisternal maturation or
  vesicle-mediated transport.

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4.10 Retention of proteins in the Golgi apparatus
depends on the membrane-spanning domain

• The membrane-spanning domain and its flanking
  sequences are sufficient to retain proteins in
  the Golgi apparatus.
• The retention mechanism for Golgi proteins
  depends on the ability to form oligomeric
  complexes and the length of the membrane-spanning
  domain.

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4.11 Rab GTPases and tethers are two types of
proteins that regulate vesicle targeting

• Monomeric GTPases of the Sar/ARF family are
  involved in generating the coat that forms
  transport vesicles.
• Another family, the Rab GTPases, are involved in
  targeting these vesicles to their destination
  membranes.

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4.11 Rab GTPases and tethers are two types of
proteins that regulate vesicle targeting

• Different Rab family members are found at each
  step of vesicle-mediated transport.
• Proteins that are recruited or activated by Rabs
  (downstream effectors) include
• tethering proteins such as long fibrous proteins
• large multiprotein complexes
• Tethering proteins link vesicles to membrane
  compartments and compartments to each other.

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4.12 SNARE proteins likely mediate fusion of
vesicles with target membranes

• SNARE proteins are both necessary and sufficient
  for specific membrane fusion in vitro, but other
  accessory proteins may be needed in vivo.
• A v-SNARE on the transport vesicle interacts with
  the cognate t-SNARE on the target membrane
  compartment.

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4.12 SNARE proteins likely mediate fusion of
vesicles with target membranes

• The interaction between v- and t-SNAREs is
  thought to bring the membranes close enough
  together so that they can fuse.
• After fusion
• the ATPase NSF unravels the v- and t-SNAREs
• the v-SNAREs are recycled to the starting
  membrane compartment

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4.13 Endocytosis is often mediated by
clathrin-coated vesicles

• The stepwise assembly of clathrin triskelions may
  help provide the mechanical means to deform
  membranes into coated pits.
• Various adaptor complexes provide the means of
  selecting cargo for transport by binding both to
• sorting signals
• clathrin triskelions

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4.13 Endocytosis is often mediated by
clathrin-coated vesicles

• GTPases of the dynamin family help release the
  coated vesicle from the membrane.
• Uncoating ATPases remove the clathrin coat before
  docking and fusion.

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4.14 Adaptor complexes link clathrin and
transmembrane cargo proteins

• Adaptor complexes bind to
• the cytoplasmic tails of transmembrane cargo
  proteins
• clathrin
• Phospholipids
• Adaptors of the AP family are heterotetrameric
  complexes of two adaptin subunits and two
  smallerproteins.

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4.14 Adaptor complexes link clathrin and
transmembrane cargo proteins

• The AP adaptors bind to sorting signals in the
  cytoplasmic tails of cargo proteins.
• The best-characterized of these signals contain
  tyrosine or dileucine residues.
• Adaptor complexes allow for the selective and
  rapid internalization of receptors and their
  ligand.

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4.15 Some receptors recycle from early endosomes
whereas others are degraded in lysosomes

• Early endosomes are mildly acidic and lack
  degradative enzymes, so
• internalized ligands can be dissociated without
  degradation of their receptors.
• Many receptors are recycled to the cell surface
  in transport vesicles that bud from the tubular
  extensions of early endosomes.

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4.15 Some receptors recycle from early endosomes
whereas others are degraded in lysosomes

• Dissociated ligands are transferred from early
  endosomes to the more acidic and hydrolase-rich
  late endosomes and lysosomes for degradation.
• Receptors that are not recycled
• are segregated into vesicles within
  multivesicular bodies
• move to late endosomes and lysosomes for
  degradation

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4.15 Some receptors recycle from early endosomes
whereas others are degraded in lysosomes

• Recycling endosomes are found adjacent to the
  nucleus.
• They contain a pool of recycling receptors that
  can be transported rapidly to the cell surface
  when needed.

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4.16 Early endosomes become late endosomes and
lysosomes by maturation

• Movement of material from early endosomes to late
  endosomes and lysosomes occurs by maturation.
• A series of ESCRT protein complexes sorts
  proteins into vesicles that bud into the lumen of
  endosomes.
• This forms multivesicular bodies that facilitate
  the process of proteolytic degradation.

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4.17 Sorting of lysosomal proteins occurs in the
trans-Golgi network

• All newly synthesized membrane and secretory
  proteins share the same pathway up until the TGN.
• There they are sorted according to their
  destinations into different transport vesicles.
• Clathrin-coated vesicles transport lysosomal
  proteins from the trans-Golgi network to maturing
  endosomes.

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4.17 Sorting of lysosomal proteins occurs in the
trans-Golgi network

• In the Golgi apparatus, mannose 6-phosphate is
  covalently linked to soluble enzymes destined for
  lysosomes.
• The mannose 6-phosphate receptor delivers these
  enzymes from the trans-Golgi network to the
  endocytic pathway.

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4.17 Sorting of lysosomal proteins occurs in the
trans-Golgi network

• Lysosomal membrane proteins are transported from
  the trans-Golgi network to maturing endosomes.
• But, they use different signals than the soluble
  lysosomal enzymes.

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4.18 Polarized epithelial cells transport
proteins to apical and basolateral membranes

• The plasma membrane of a polarized cell has
  separate domains with distinct sets of proteins.
• This necessitates a further sorting step.
• Depending on the cell type, sorting of cell
  surface proteins in polarized cells can occur at
• the TGN
• endosomes
• one of the plasma membrane domains
• Sorting in polarized cells is mediated by
  specialized adaptor complexes and perhaps lipid
  rafts and lectins.

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4.19 Some cells store proteins for later secretion

• Some cargo molecules are stored in secretory
  granules, which
• fuse with the plasma membrane
• release their contents only upon stimulation
• Storage of proteins for regulated secretion often
  involves a condensation process.
• Cargo self-associates, condensing to form a
  concentrated packet for eventual delivery to the
  outside of the cell.

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4.19 Some cells store proteins for later secretion

• Condensation of proteins for regulated secretion
  often
• begins in the endoplasmic reticulum
• continues in the Golgi apparatus
• is completed in condensing vacuoles that finally
  yield secretory granules
• Condensation is accompanied by selective membrane
  retrieval at all stages of exocytosis.

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4.19 Some cells store proteins for later secretion

• Fusion of synaptic vesicles with the plasma
  membrane involves SNARE proteins.
• But it is regulated by calcium-sensitive proteins
  such as synaptotagmin.