Chapter 4

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. 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.

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

5. 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 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.

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. 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.

8. 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.

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. 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.

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. 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.

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. 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.

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. 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.

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. 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.

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. 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.

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. 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

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. 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.

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. 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.

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. 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

32. 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.

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. 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.

36. 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.

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. 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.

40. 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.