Chapter 4 Protein trafficking between membranes By Graham Warren & Ira Mellman.

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Chapter 4 Protein trafficking between membranes By Graham Warren & Ira Mellman

Transcript of Chapter 4 Protein trafficking between membranes By Graham Warren & Ira Mellman.

Page 1: Chapter 4 Protein trafficking between membranes By Graham Warren & Ira Mellman.

Chapter 4

Protein trafficking between membranesBy

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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• 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.

4.1 Introduction

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• The vesicles form by budding from an organelle’s surface.

• They subsequently fuse with the target membrane of the acceptor compartment.

4.1 Introduction

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• 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 protein’s amino acid sequence or carbohydrate structures.

• Transport vesicles contain proteins that target them specifically to their intended destinations with which they dock and fuse.

4.1 Introduction

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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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• 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.

4.2 Overview of the exocytic pathway

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• 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.

4.2 Overview of the exocytic pathway

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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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• 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.

4.4 Concepts in vesicle-mediated protein transport

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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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• 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.

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

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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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• 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.

4.6 COPII-coated vesicles mediate transport from the ER to 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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• 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.

4.7 Resident proteins that escape from the ER are retrieved

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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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• 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.

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

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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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• 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.

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

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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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• 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

4.12 SNARE proteins likely mediate fusion of vesicles with target membranes

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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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• GTPases of the dynamin family help release the coated vesicle from the membrane.

• Uncoating ATPases remove the clathrin coat before docking and fusion.

4.13 Endocytosis is often mediated by clathrin-coated vesicles

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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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• 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.

4.14 Adaptor complexes link clathrin and transmembrane cargo proteins

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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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• 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

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

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• 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.

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

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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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• 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.

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

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

lysosomal enzymes.

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

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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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• 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.

4.19 Some cells store proteins for later secretion

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

synaptotagmin.

4.19 Some cells store proteins for later secretion