This chapter discusses protein trafficking between organelle membranes in eukaryotic cells. It describes the core exocytic and endocytic pathways involving transport of cargo proteins between compartments via transport vesicles. Key concepts covered include vesicle coating by COPI and COPII, the roles of Rab GTPases and SNAREs in vesicle targeting and fusion, and mechanisms of sorting cargo into transport vesicles and retaining resident proteins within organelles. The chapter also discusses specialized processes in polarized cells and regulated secretion.

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

2. 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

3. • 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

4. • The vesicles form by budding from an organelle’s
surface.
• They subsequently fuse with the target membrane
of the acceptor compartment.
4.1 Introduction

5. • 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

6. 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

7. • 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

8. • 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

9. 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.

10. 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

11. • 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

12. 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.

13. • 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

14. 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.

15. • 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

16. 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.

17. • 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

18. 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.

19. • 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

20. 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.

21. 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.

22. 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.

23. • 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

24. 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.

25. • 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

26. 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

27. • 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

28. 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.

29. • 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

30. 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.

31. • 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

32. • 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

33. 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.

34. 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.

35. • 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

36. • 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

37. 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.

38. 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.

39. • 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

40. • 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