Intracellular Vesicular Traffic The molecular mechanisms of membrane transport and the maintenance of compartment diversity Transport from the ER through the Golgi apparatus Transport from the trans Golgi network to lysosomes Transport into the cell from the plasma membrane: endocytosis (Discussion topic) Transport from the trans Golgi network to the cell exterior: exocytosis Chapter 13 Molecular Biology of the Cell Copyright © Garland Science 2008

Vesicular transport: simultaneous transport of soluble components and membrane components. fusion budding Note: Both fusion and budding are energy consuming processes. Figure 13-1,2 Molecular Biology of the Cell (© Garland Science 2008)

The “road map” of vesicular transport export import recycle

The molecular mechanisms of membrane transport and the maintenance of compartment diversity There are various types of coated vesicles. The assembly of a clathrin coat drives vesicle formation. Not all coats form a basketlike structures. Cytoplasmic proteins regulate the pinching-off and uncoating of coated vesicles. Monomeric GTPases control coat assembly. Not all transport vesicles are spherical. Rab proteins guide vesicle targeting. SNAREs mediate membrane fusion. Interacting SNAREs need to be pried apart before they can function again. Viral fusion proteins and SNAREs may use similar fusion mechanisms.

Three major types of coated vesicles Golgi Coating protein: COP (nickname for police) Figure 13-4 Molecular Biology of the Cell (© Garland Science 2008)

The use of different (at least 3) coats in vesicular transport Retromer Coat proteins are located in the cytosol to help vesicle budding and cargo collection.

Clathrin-coated vesicles The cytoplasmic side of the plasma membrane Figure 13-7c, d Molecular Biology of the Cell (© Garland Science 2008)

Figure 13-7a, b Molecular Biology of the Cell (© Garland Science 2008)

The assembly and disassembly of a clathrin coat during cargo collection and vesicle formation. Figure 13-8 Molecular Biology of the Cell (© Garland Science 2008)

Dynamin and associated proteins regulate the pinching-off of clathrin-coated vesicles. Figure 13-12a Molecular Biology of the Cell (© Garland Science 2008)

another kind of coating complex Retrograde transport from endosomal membranes to Golgi is mediated by retromer proteins (SNX1, VPS29,35, 26). Retromer proteins: another kind of coating complex A retromer coat is assembled only when the complex binds to the cytoplasmic tails of cargo receptors; interacts with a curved lipid bilayer (vesicle); and binds to PI(3)P, the endosomal marker. Figure 13-9 Molecular Biology of the Cell (© Garland Science 2008)

Various phosphoinositides serve as protein binding sites and mark organelles and membrane domains. Inositol PI-associated vesicular transport proteins Figure 13-10 Molecular Biology of the Cell (© Garland Science 2008)

PIs mark organelles and membrane domains PI(5)P phosphatase PI 5-kinase Figure 13-11 Molecular Biology of the Cell

Directed and selective transport of particular components from one membrane-enclosed component to another maintains the differences between those compartments. The assembly of the coat helps not only to drive the formation of the vesicle, but to collect specific membrane and soluble cargo molecules for transport. Clathrin-coated vesicles mediate transport from the plasma membrane and the trans Golgi network; COPI- and COPII-coated vesicles mediate transport between Golgi cisternae and between the ER and the Golgi apparatus, respectively. Adaptor proteins link the clathrin to the vesicle membrane and also trap specific cargo molecules. The coat is shed rapidly after budding to facilitate vesicle fusion with target membrane. Retromer-coated vesicles mediate transport from endosomal membranes to the trans Golgi network.

Budding: use COPs (with the help of monomeric GTPases) to form vesicles (造「飛碟」啟程)

Coat-recruitment monomeric GTPases control coat assembly/disassembly. Arf proteins help COPI coat assembly and clathrin coat assembly at Golgi. Sar1 protein help COPII coat assembly at ER (as shown here as an example). Sec23/24: COPII proteins (inner coat) (Guanine nucleotide Exchange Factor) Figure 13-13a,b Molecular Biology of the Cell (© Garland Science 2008)

The formation of COPII-coated vesicles. Sec13/31: COPII proteins (outer coat) Sar1-GDP 脫殼 Sar1-GTP hydrolysis accelerates COPII coat disassembly. Figure 13-13c,d Molecular Biology of the Cell (© Garland Science 2008)

Targeting: use Rabs and Rab receptors to find the target (航向目的地)

Rab proteins guide vesicle targeting. Table 13-1 Molecular Biology of the Cell (© Garland Science 2008)

Rab-dependent tethering of a vesicle to a target membrane. (拴住) (停靠) (融合) Figure 13-14 Molecular Biology of the Cell (© Garland Science 2008)

The formation of a Rab5 domain on the endosome membrane. Cytoplasmic side [binds to PI(3)P] Endosome Figure 13-15 Molecular Biology of the Cell (© Garland Science 2008)

Fussion: use v-SNARs and t-SNAREs to join membranes (連船帶貨一同點交)

SNAREs mediate membrane fusion: A trans-SNARE complex. (>35 fusion proteins) Figure 13-16 Molecular Biology of the Cell (© Garland Science 2008)

SNAREs mediate membrane fusion Figure 13-17 Molecular Biology of the Cell (© Garland Science 2008)

Dissociation of SNARE pairs by NSF, a post-fusion ATP-consuming process. Interacting SNAREs need to be pried apart before they can function again. The v-SNARE might return to the donor compartment for future usage. Figure 13-18 Molecular Biology of the Cell (© Garland Science 2008)

Viral fusion proteins and SNAREs may use similar fusion mechanisms. Figure 13-19a Molecular Biology of the Cell (© Garland Science 2008)

The fusion of HIV with T-cell Viral Gp 120 mediates targeting and facilitates the insertion of viral fusion protein. CD4+ T-cell (引狼入室的過程) Figure 13-19b Molecular Biology of the Cell (© Garland Science 2008)

Local synthesis of phosphoinositides creates protein binding sites that trigger coat assembly and vesicle budding. Monomeric GTPases help regulate vesicle budding and ducking. Sar1 and Arf proteins regulate coat assembly and disassembly, whereas Rab proteins function as vesicle targeting GTPases. GTP binding and hydrolysis dynamically control the assembly and disassembly of Rab proteins and their effector proteins (including motor proteins that travel along the cytoskeletal network and tethering proteins that help to dock vesicles to the target membrane). Complementary v-SNARE proteins on transport vesicles and t-SNARE proteins on the target membrane form stable trans-SNARE complexes to help the fusion of lipid bilayers.

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Transport from the ER through the Golgi apparatus Only proteins that are properly folded and assembled can leave the ER. Vesicular tubular clusters mediate transport from the ER to the Golgi apparatus. The retrieval pathway to the ER uses sorting signals Many proteins are selectively retained in the compartments in which they function. The Golgi apparatus consists of an ordered series of compartments. Oligosaccharide chains are processed in the Golgi apparatus. Proteoglycans are assembled in the Golgi apparatus. What is the purpose of glycosylation? Transport through the Golgi apparatus may occur by vesicular transport or by cisternal maturation. Golgi matrix proteins help organize the stack.

Only proteins that are properly folded and assembled can leave the ER. Calnexin, calretilculin, and ERp57 are chaperones. Incorrect Correct Remember (Ch. 12): Only proteins that are properly folded and assembled can leave the ER. Figure 12-53 Molecular Biology of the Cell (© Garland Science 2008)

Proteins are retained in the ER until they are properly assembled. Bip: a chaperone protein which also prevents the pre-mature exit of proteins. Figure 13-21 Molecular Biology of the Cell (© Garland Science 2008)

Properly folded and assembled proteins leave the ER in COPII-coated vesicles. With the help from ER exit signals Figure 13-20 Molecular Biology of the Cell (© Garland Science 2008)

Transport vesicles form vesicular tubular clusters by homotypic fusion before fussing with cis Golgi network. Homotypic fusion: both vesicles contain the same set of t-SNAREs and v-SNAREs. Remember the function of NSF? Figure 13-22 Molecular Biology of the Cell (© Garland Science 2008)

Figure 13-23a Molecular Biology of the Cell (© Garland Science 2008)

What for? Figure 13-23b Molecular Biology of the Cell (© Garland Science 2008)

Escaped ER resident proteins are captured by KDEL receptors and returned by COPI-coated vesicles (along with membrane components). Lys-Asp-Glu-Leu (KDEL) is an ER retention signal. Figure 13-24a Molecular Biology of the Cell (© Garland Science 2008)

cis medial trans Figure 13-24b Molecular Biology of the Cell (© Garland Science 2008)

The Golgi apparatus Figure 13-25b Molecular Biology of the Cell (© Garland Science 2008)

The Golgi apparatus consists of an ordered series of compartments (cisternae). Figure 13-25a Molecular Biology of the Cell (© Garland Science 2008)

Molecular compartmentalization of the Golgi apparatus. Cis-Golgi Osmium Trans-Golgi Nucleoside di-phosphatase TGN Acid phosphatase Figure 13-27 Molecular Biology of the Cell (© Garland Science 2008)

Intestinal lumen A goblet cell of the small intestine contains a polarized Golgi apparatus and large numbers of secretory vesicles for unidirectional secretion into the intestinal lumen. Figure 13-29 Molecular Biology of the Cell (© Garland Science 2008)

Oligosaccharides are processed in the Golgi apparatus. ER Subdivisions of the Golgi apparatus are functionally differentiated. Figure 13-28 Molecular Biology of the Cell (© Garland Science 2008)

Glycosylation of proteins. Figure 13-32 Molecular Biology of the Cell (© Garland Science 2008)

Two main classes of N-linked oligosaccharides are found in mature mammalian glycoproteins. Endo-H resistant Endo-H sensitive Figure 13-30 Molecular Biology of the Cell (© Garland Science 2008)

Oligosaccharide processing in the ER and the Golgi apparatus. Figure 13-31 Molecular Biology of the Cell (© Garland Science 2008)

Hypothetic roles of glycosylation? Figure 13-34 Molecular Biology of the Cell (© Garland Science 2008)

Transport through the Golgi apparatus may occur by vesicular transport or by cisternal maturation. What purpose do the retrieval processes serve for? Figure 13-35 Molecular Biology of the Cell (© Garland Science 2008)

Correctly folded and assembled proteins in the ER are packaged into COPII-coated transport vesicles that shed their coat and fuse with one another to form vesicular tubular clusters. The clusters then move on microtubule tracks to the Golgi apparatus. Any resident ER proteins that escape from the ER are returned there from the vesicular tubular clusters and Golgi by retrograde transport in COPI-coated vesicles. The cis, medial, and trans cisternae of Golgi apparatus contain many sugar nucleotides and glycosyl transferases to perform glycosylation reactions on lipid and protein molecules as they pass through. Continual retrograde vesicular transport from more distal cisternae is thought to keep the enzymes concentrated in the cisternae where they are needed. The finished new proteins end up in the trans Golgi network (TGN), which packages them in transport vesicles and dispatches them to their specific destinations in the cell.

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Transport from the trans Golgi network to lysosomes Lysosomes are the principal sites of intracellular digestion. Lysosomes are heterogeneous. Plant and fungal vacuoles are remarkably versatile lysosomes. Multiple pathways deliver materials to lysosomes. A mannose-6-phosphate receptor recognizes lysosomal proteins in the trans Golgi network. The M6p receptor shuttles between specific membranes. A signal patch in the hydrolase polypeptide chain provides the cue for M6p addition. Defects in the GlcNAc phosphotransferase cause a lysosomal storage disease in humans. Some lysosomes undergo exocytosis.

Figure 13-36 Molecular Biology of the Cell (© Garland Science 2008)

Vesicles carrying acid hydrolases from TGN Lysosomes can be visualized by labeling the product of its marker enzyme, acid phosphatase. Figure 13-37 Molecular Biology of the Cell (© Garland Science 2008)

Lysosomes are heterogeneous. Note: Plant and fungal vacuoles are versatile lysosomes. Figure 13-38 Molecular Biology of the Cell (© Garland Science 2008)

Three pathways to degradation in lysosomes. Figure 13-42 Molecular Biology of the Cell (© Garland Science 2008)

Autophagy: a lysosome-dependent organelle degradation process. What purpose does autophagy serve? Figure 13-41 Molecular Biology of the Cell (© Garland Science 2008)

Example Oligosaccharides are processed in the Golgi apparatus. ER Example Subdivisions of the Golgi apparatus are functionally differentiated. Figure 13-28 Molecular Biology of the Cell (© Garland Science 2008)

Lysosomal hydrolases are tagged by a M6P group in Golgi stacks, sorted in TGN, and finally transported to lysosomes. Sorting Transport Tagging Targeting/fusion Sorting Transport Targeting/fusion Figure 13-44 Molecular Biology of the Cell (© Garland Science 2008)

Recognized by M6P receptors for sorting to lysosomes Lysosomal hydrolase (a soluble enzyme) signal patch Figure 13-43 Molecular Biology of the Cell (© Garland Science 2008)

The recognition of a lysosomal hydrolase. The signal patch is recognized by GlcNAc phosphotransferase in the Golgi lumen. A special type of protein glycosylation (M6P) serves as the final sorting signal for vesicular transport to lysosomes. Figure 13-45 Molecular Biology of the Cell (© Garland Science 2008)

Q: I-cell (inclusion-cell) disease is a severe form of lysosomal storage disease; almost all of the hydrolytic enzymes are missing from the lysosomes and end up in the blood. Their undigested substrates accumulate in lysosomes, which form large inclusions in the patients’ cells. What could be the underlying causes of this disease?

Lysosomes contain unique membrane proteins and a wide variety of soluble hydrolytic enzymes that operate under acidic conditions, which is maintained by an ATP-driven lysosomal H+ pump. The lysosomal hydrolases contain N-linked oligosaccharides that are covalently modified in an unique way in the cis Golgi network so that their mannose residues are phosphorylated to become mannose-6-phosphate (M6P). M6P is recognized by the M6P receptor in the trans Golgi network that segregates the hydrolases and helps package them into budding transport vesicles to be delivered to endosomes. The low pH in endosomes and removal of the phosphate from the M6P cause the dissociation of hydrolases from M6P receptors; and the latter shuttle back to TGN. Another M6P-independent transport system uses clathrin-coated vesicles to deliver resident lysosomal membrane proteins from the TGN.

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Transport from the trans Golgi network to the cell exterior: exocytosis Many proteins and lipids seem to be carried automatically from the Golgi apparatus to the cell surface. Secretory vesicles bud from the trans Golgi network. Proteins are often proteolytically processed during the formation of secretory vesicles. Secretory vesicles wait near the plasma membrane until signaled to release their contents. Regulated exocytosis can be a localized response of the plasma membrane and its underlying cytoplasm. Secretory vesicle membrane components are quickly removed from the plasma membranes. Some regulated exocytosis events serve to enlarge the plasma membrane. Polarized cells direct proteins from the trans Golgi network to the appropriate domain of the plasma membrane. Different strategies guide membrane proteins and lipids selectively to the correct plasma membrane domains. Synaptic vesicles can form directly from endocytic vesicles.

The constitutive and regulated secretory pathways. Figure 13-63 Molecular Biology of the Cell (© Garland Science 2008)

Protein sorting pathways in TGN. Figure 13-64 Molecular Biology of the Cell (© Garland Science 2008)

Immature secretory vesicle (with clathrin) (without clathrin) Gold particle-conjugated anti-clathrin antibody Figure 13-65b Molecular Biology of the Cell (© Garland Science 2008)

The formation of secretory vesicles. (increasing acidity and partial membrane retrieval) Figure 13-65a Molecular Biology of the Cell (© Garland Science 2008)

The exocytosis of secretory vesicles. Figure 13-66 Molecular Biology of the Cell (© Garland Science 2008)

Proteins are often proteolytically processed during the formation of secretory vesicles. For transport into ER Figure 13-67 Molecular Biology of the Cell (© Garland Science 2008)

Exocytosis in mast cells. Figure 13-68 Molecular Biology of the Cell (© Garland Science 2008)

Exocytosis can be a localized event, indicating that a plasma membrane segment and its underlying cytoplasm can function independently. Figure 13-69 Molecular Biology of the Cell (© Garland Science 2008)

Some regulated exocytosis events serve to enlarge the plasma membrane. Under most conditions, secretory vesicle membrane components are rapidly removed form the plasma membrane by endocytosis. Figure 13-70 Molecular Biology of the Cell (© Garland Science 2008)

Polarized cells must have some means to generate and maintain their plasma membrane domains. Even intracellular organelles that are connected together (examples?) must be able to generate and maintain their membrane domains. Figure 13-71 Molecular Biology of the Cell (© Garland Science 2008)

Sorting of plasma membrane proteins in polarized cells. Figure 13-72 Molecular Biology of the Cell (© Garland Science 2008)

The formation of synaptic vesicles. Glutamate transporter Figure 13-73 Molecular Biology of the Cell (© Garland Science 2008)

A synaptic vesicle 70 copies 1~2 copies 1800x Figure 13-74 Molecular Biology of the Cell (© Garland Science 2008)

Cells can secrete molecules by exocytosis in either a constitutive or a regulated fashion. In the regulated pathways, the molecules are stored either in secretory vesicles or in synaptic vesicles, which do not fuse with the plasma membrane until a signal is provided. Secretory vesicles bud from TGN with secretory proteins being concentrated during their maturation. Synaptic vesicles form from both endocytic vesicles and from endosomes, and they mediate the secretion of neurotransmitters. Proteins are delivered from the TGN to the plasma membrane by the constitutive pathway unless they are diverted into other pathways or retained in the Golgi apparatus. In polarized cells, the TGN to plasma membrane pathways operate selectively to ensure that different sets of membrane components and secreted proteins are delivered to the designated domains of the plasma membrane.

Thank you!