Lecture 5

Intracellular Vesicular Transport

Chapter 13 MBoC (5th Edition) Alberts et al.

2/15/12

Copyright © Garland Science1 2008 A Simplified “Roadmap” of Protein Traffic

Proteins can move from one compartment to another by gated transport (red), transmembrane transport (blue), or vesicular transport (green). The sorting signals that direct a given protein’s movement through the system, and thereby determine its eventual location in the cell, are contained in each protein’s amino acid sequence. The journey begins with the synthesis of a protein on a ribosome in the cytosol and terminates when the protein reaches its final destination. At each intermediate station (boxes), a decision is made as to whether the protein is to be retained in that compartment or transported further. In principle, a sorting signal could be required for either retention in or exit from a compartment.

Figure 12-6 2 Intracellular Vesicular Traffic

Exocytosis and Endocytosis

Figure 13-1

(A) In exocytosis, a transport vesicle fuses with the plasma membrane. Its content is released into the extracellular space, while the vesicle membrane (red) becomes continuous with the plasma membrane. (B) In endocytosis, a plasma membrane patch (red) is internalized forming a transport vesicle. Its content derives from the extracellular space.

3 Vesicular Transport

Figure 13-2

Transport vesicles bud off from one compartment and fuse with another. As they do so, they carry material as cargo from the lumen (the space within a membrane-enclosed compartment) and membrane of the donor compartment to the lumen and membrane of the target compartment. The processes of vesicle budding and fusion are not symmetrical: budding requires a membrane fusion event initiated from the lumenal side of the membrane, while vesicle fusion requires a membrane fusion event initiated from the cytoplasmic side of both donor and target membranes. 4 The Intracellular Compartments of the Eucaryotic Cell Involved in the Biosynthetic-Secretory and Endocytic Pathways

Figure 13-3 *Retrieval pathways are shown with blue arrows.

In the endocytic and exocytic pathways, cargo proteins are transferred between compartments by transport vesicles. 5 Different Coats Are Utilized in Vesicular Transport

Three well-characterized types of coated vesicles: each type is used for different transport steps.

Figure 13-4

Different coat proteins select different cargo and shape the transport vesicles that mediate the various steps in the biosynthetic– secretory and endocytic pathways. Figure 13-5 6 The Assembly and Disassembly of a Clathrin Coat

Figure 13-8

The assembly of the coat introduces curvature into the membrane, which leads to the formation of uniformly sized coated buds. The adaptor proteins bind clathrin triskelions and membrane-bound cargo receptors, thereby mediating the selective recruitment of both membrane and cargo molecules into the vesicle. The clathrin coat is rapidly lost shortly after the vesicle forms. 7 The Structure of Clathrin Coat

EM of clathrin triskelion with platinum. Each triskelion is composed of three clathrin heavey chains and three clathrin light chains.

Figure 13-6 EM shows clathrin-coated pits Figure 13-7 and veiscles on the inner surface A cryo-EM taken of a clathrin coat of the plasma membrane of composed of 36 triskelions organized in a cultured fibroblasts. network of 12 pentagons and 6 hexagons, with heavy chains (left) and light chains (right) highlighted. 8 The Role of Dynamin in Pinching off Clathrin-Coated Vesicles

The dynamin assembles into a ring around the neck of the forming bud. The dynamin ring is thought to recruit other proteins to the vesicle neck, which, together with dynamin, destabilize the interacting lipid bilayers so that the noncytoplasmic leaflets flow together. The newly formed vesicle then pinches off from the membrane. Specific mutations in dynamin can either enhance or block the pinching- off process.

A thin-section EM: Deeply invaginated clathrin-coated pits form in the fly’s nerve cells, with a ring of mutant dynamin assembled around the neck. The pinching-off process fails because membrane fusion does not take place. Figure 13-12 9 Rab Proteins Guide Vesicle Targeting

Vesicle targeting depends on two types of proteins: - Rab proteins (GTPases) direct the vesicle to specific spots on the correct target membrane by interacting with Rab effectors; - SNARE proteins mediate the fusion of the lipid bilayers. Rab effector proteins interact via active Rab proteins (Rab-GTPs, yellow) located on the target membrane, vesicle membrane, or both, to establish the first connection between the two membranes that are going to fuse. In the example shown here, the Rab effector is a filamentous tethering protein (green). Next, SNARE proteins (v- and t-SNAREs) on the two membranes (red and blue) pair to dock the vesicle to the target membrane and catalyze the fusion of the two apposed lipid bilayers.

Figure 13-14

10 Interacting SNAREs Need to Be Separated Before They Can Function Again

Figure 13-18

Dissociation of SNARE pairs by NSF after a membrane fusion cycle. After a v-SNARE and t-SNARE have mediated the fusion of a transport vesicle on a target membrane, the NSF (ATPase) binds to the SNARE complex and, with the help of two accessory proteins, hydrolyzes ATP to pry the SNAREs apart.

11 Rab Proteins Help Ensure the Specificity of Vesicle Docking

Rabs: GTPases. Each Rab has a characteristic distribution on cell membranes and every organelle has at least one Rab protein on its cytosolic surface. Proteins (downstream effectors) that are recruited or activated by Rabs include: - tethering proteins such as long fibrous proteins - large multiprotein complexes Tethering proteins link vesicles to membrane compartments and compartments to each other.

12 Transport from the ER to the Golgi Apparatus

13 The Recruitment of Cargo Molecules into ER Transport Vesicles

By binding directly or indirectly to the COPII coat, membrane and soluble cargo proteins, respectively, become concentrated in the transport vesicles as they leave the ER. Figure 13-20

Membrane proteins are packaged into budding transport vesicles through interactions of exit signals on their cytosolic tails with the COPII coat. Some of the membrane proteins that the coat traps function as cargo receptors, binding soluble proteins in the lumen and helping to package them into vesicles. A typical 50-nm transport vesicle contains about 200 membrane proteins, which can be of many different types. As indicated, unfolded or incompletely assembled proteins are bound to chaperones and retained in the ER compartment. 14 A Model for the Formation of a COPII-Coated Vesicle

A complex of two additional Sar1: a coat COPII coat proteins, called recruitment Sec13/31, forms the outer GTPase shell of the coat.

Membrane bound, active Sar1-GTP recruits COPII subunits to the membrane, causing the membrane to form a bud, which includes selected transmembrane proteins. A subsequent membrane-fusion event pinches off the coated vesicle. GTP-bound Sar1 binds to a complex of two COPII coat proteins, called Sec23/24. 15 Figure 13-13 Vesicular Tubular Clusters

(B) Figure 13-23 (A)

(A) An EM section of vesicular tubular clusters forming from the ER membrane. Many of the vesicle-like structures seen in the micrograph are cross sections of tubules that extend above and below the plane of this thin section and are interconnected. (B) Vesicular tubular clusters move along microtubules to carry proteins from the ER to the Golgi apparatus. COPI coats mediate the budding of vesicles that return to the ER from these clusters. 16 A Model for the Retrieval of Soluble ER Resident Proteins

Figure 13-24 (A) (B) ER resident proteins that escape from the ER are returned by vesicular transport. (A) The KDEL receptor present in vesicular tubular clusters and the Golgi apparatus captures the soluble ER resident proteins and carries them in COPI-coated transport vesicles back to the ER. Upon binding its ligands in this environment, the KDEL receptor may change conformation, so as to facilitate its recruitment into budding COPI-coated vesicles. (B) The retrieval of ER proteins begins in vesicular tubular clusters and continues from all parts of the Golgi apparatus. In the environment of the ER, the ER resident proteins dissociate from the KDEL receptor, which is then returned to the Golgi apparatus for reuse. 17 The Golgi Apparatus Consists of an Ordered Series of Compartments (A) 3-D reconstruction from EMs of the Golgi apparatus in a secretory animal cell. The cis face of the Golgi stack is that closest to the ER. (B) A thin section EM emphasizing the transitional zone between the ER and the Golgi apparatus in an animal cell. (C) An EM of a Golgi apparatus in a plant cell (the green alga Chlamydomonas) seen in cross section. In plant cells, the Golgi apparatus is generally more distinct and more clearly separated from other intracellular membranes than in animal cells. (A)

(C) 18

(B) Figure 13-25 The Functional Compartmentalization of the Golgi Apparatus

Oligosaccharide processing in Golgi compartments. Processing enzymes are not restricted to a particular cisterna; instead, their distribution is graded across the stack— such that early-acting enzymes are present mostly in the cis Golgi cisternae and later-acting enzymes are mostly in the trans Golgi cisternae.

Figure 13-28 19 Oligosaccharide Processing in the ER and the Golgi Apparatus

Figure 13-31

Endo H: a highly specific endoglycosidase. Two broad classes of N-linked oligosaccharides, the complex oligosaccharides and the high-mannose oligosaccharides, are attached to mammalian glycoproteins. Sometimes, both types are attached, in different places, to the same polypeptide. The oligosaccharide processing pathway is highly ordered, so that each step shown depends on the previous one. 20 Models for Transport Through the Golgi Apparatus

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

21 Figure 13-35 Transport into the Cell from the Plasma Membrane: Endocytosis

Two main types of endocytosis differ according to the size of the endocytic vesicles formed.

Phagocytosis (“cell eating”): large particles are ingested via large vesicles called phagosomes (generally >250 nm in diameter).

Pinocytosis (“cell drinking”): fluid and solutes are ingested via small pinocytic vesicles (about 100 nm in diameter).

22 Cells Use Receptor-Mediated Endocytosis to Import Selected Extracellular Macromolecules

A low-density lipoprotein (LDL) particle. It contains a core of about 1500 cholesterol molecules esterified Figure 13-51 to long-chain fatty acids. A lipid Normal (A) and mutant (B) LDL receptors. monolayer composed of about 800 A mutant cell in which the LDL receptors are abnormal and lack the phospholipid and 500 unesterified site in the cytoplasmic domain that enables them to bind to adaptor cholesterol molecules surrounds the proteins in the clathrin-coated pits. Such cells bind LDL but cannot core of cholesterol esters. A single ingest it. In most human populations, 1 in 500 individuals inherits molecule of a 500 kDa protein one defective LDL receptor gene and, as a result, has an increased organizes the particle and mediates risk of a heart attack caused by atherosclerosis. 23 the specific binding of LDL to cell- surface LDL receptors. The Receptor-Mediated Endocytosis of LDL

Figure 13-53

The low-density lipoprotein (LDL) dissociates from its receptors in the acidic environment of the early endosome. After a number of steps, the LDL ends up in lysosomes, where it is degraded to release free cholesterol. In contrast, the LDL receptors are returned to the plasma membrane via clathrin-coated transport vesicles that bud off from the tubular region of the early endosome. Whether it is occupied or not, an LDL receptor typically makes one round trip into the cell and back to the plasma membrane every 10 minutes, making a total of several hundred 24 trips in its 20-hour lifespan. Storage of Plasma Membrane Proteins in Recycling Endosomes

Figure 13-61 Recycling endosomes can serve as an intracellular pool of specialized plasma membrane proteins that can be mobilized when needed. In the example shown, insulin binding to the insulin receptor triggers an intracellular signaling pathway that causes the rapid insertion of glucose transporters into the plasma membrane of a fat or muscle cell, greatly increasing glucose intake. 25 Transport from the Trans Golgi Network to the Cell Exterior — Exocytosis

The fusion of the vesicles with the plasma membrane is called exocytosis. Transport vesicles destined for the plasma membrane normally leave the TGN in a steady stream as irregularly shaped tubules. The membrane proteins and the lipids in these vesicles provide new components for the cell’s plasma membrane, while the soluble proteins inside the vesicles are secreted to the extracellular space.

26 Three Best-Understood Pathways of Protein Sorting in the Trans Golgi Network

Figure 13-64

(1) Proteins with the mannose 6-phosphate (M6P) marker are diverted to lysosomes (via endosomes) in clathrin-coated transport vesicles. (2) Proteins with signals directing them to secretory vesicles are concentrated in such vesicles as part of a regulated secretory pathway that is present only in specialized secretory cells. (3) In unpolarized cells, a constitutive secretory pathway delivers proteins with no special features to the cell surface. 27 The Formation of Secretory Vesicles

Figure 13-65 (A) Secretory proteins become segregated and highly concentrated in secretory vesicles by two mechanisms: 1) they aggregate in the ionic environment of the trans Golgi network; often the aggregates become more condensed as secretory vesicles mature and their lumen becomes more acidic. 2) clathrin-coated vesicles retrieve excess membrane and lumenal content present in immature secretory vesicles as the secretory vesicles mature. (B) This EM shows secretory vesicles forming from the trans Golgi network in an insulin secreting β cell of the pancreas. An antibody conjugated to gold spheres (black dots) has been used to locate clathrin molecules. The immature secretory vesicles (open arrow), which contain insulin precursor protein (proinsulin), contain clathrin patches. Clathrin coats are no longer seen on the mature secretory vesicle, which has a highly condensed core (solid arrow). 28 Transport from the Trans Golgi Network to Lysosomes

29 Lysosomes Are the Principal Sites of Intracellular Digestion

Histochemical visualization of lysosomes. These EMs show two sections of a cell stained to reveal the location of acid phosphatase, a marker enzyme for lysosomes. The larger membrane enclosed organelles, containing dense precipitates of lead phosphate, are lysosomes.

Figure 13-36

The acid hydrolases are hydrolytic enzymes that Figure 13-38 are active under acidic A model for lysosome maturation. The heterogeneity of conditions. A V-type ATPase in lysosomal morphology reflects, in part, the different nature the membrane pumps H+ into of the materials delivered to the organelle, as well as the the lysosome, maintaining its different stages in the maturation cycle. 30 lumen at an acidic pH. Transport of Newly Synthesized Lysosomal Hydrolases to Lysosomes

Figure 13-44

The sequential action of two enzymes in the cis and trans Golgi network adds mannose 6-phosphate (M6P) groups to the precursors of lysosomal enzymes. They then segregate from all other types of proteins in the TGN because monomeric adaptor proteins in the clathrin coat bind the M6P receptors, which, in turn, bind the modified lysosomal hydrolases. The clathrin-coated vesicles bud off from the TGN, shed their coat, and fuse with early endosomes. At the lower pH of the endosome, the hydrolases dissociate from the M6P receptors, and the empty receptors are recycled in retromer-coated vesicles to the Golgi apparatus for further rounds of transport. In the endosomes, the phosphate is removed from the mannose sugars attached to the hydrolases, further ensuring that the hydrolases do not return 31 to the Golgi apparatus with the receptor. Three Pathways to Degradation in Lysosomes

Figure 13-42

(A) Materials in each pathway are derived from a different source. Note that the autophagosome has a double membrane. (B) An EM of an autophagosome containing a mitochondrion and a peroxisome. 32 Multiple Pathways Deliver Materials to Lysosomes

Figure 13-41 A model of autophagy. After a nucleation event in the cytoplasm, a crescent of autophagosomal membrane grows by fusion of vesicles of unknown origin that extend its edges. Eventually, a membrane fusion event closes the autophagosome, sequestering a portion of the cytoplasm of the cell in a double membrane. The autophagosome then fuses with lysosomes containing acid hydrolases that digest its content.

33 The Endocytic Pathway from the Plasma Membrane to Lysosomes

Maturation of early endosomes to late endosomes occurs through the formation of multivesicular bodies, which contain large amounts of invaginated membrane and internal vesicles. Multivesicular bodies move inward along microtubules, continually shedding transport vesicles that recycle components to the plasma membrane. They gradually convert into late endosomes, either by fusing with each other or by fusing with preexisting late endosomes. The late endosomes no longer send vesicles to the plasma membrane.

Figure 13-56 34 The Sequestration of Endocytosed Proteins into Internal Membranes of Multivesicular Bodies

Eventually, proteases and lipases in lysosomes digest all of the internal membranes within multivesicular bodies produced by the invaginations. The invagination processes are essential to achieve complete digestion of endocytosed membrane proteins: because the outer membrane of the multivesicular body becomes continuous with the lysosomal membrane, for example, lysosomal hydrolases could not digest the cytosolic domains of endocytosed transmembrane proteins such as the EGF receptor shown here, if the protein were not localized in internal vesicles.

Figure 13-57 35 The Entry of Enveloped Viruses (e.g. HIV) into Cells

(A)

(B)

Figure 13-19

(A) EMs showing how HIV enters a cell by fusing its membrane with the plasma membrane of the cell. (B) A model for the fusion process. HIV binds first to the CD4 protein on the surface of the target cell. The viral gp120 protein, which is bound to the HIV fusion protein, mediates this interaction. A second cell-surface protein on the target cell, chemokines receptor, interacts with gp120. This interaction releases the HIV fusion protein from gp120, allowing the previously buried hydrophobic fusion peptide to insert into the plasma membrane of the target cell. The fusion protein, which is a trimer, thus becomes transiently anchored as an integral membrane protein in the two opposing membranes. The fusion protein then spontaneously rearranges, collapsing into a tightly packed six-helix bundle. The energy released by this rearrangement in multiple copies of the fusion protein pulls the two membranes together, overcoming the high activation energy barrier that normally prevents membrane fusion. Thus, like a mouse trap, the HIV fusion protein contains a reservoir of potential energy, which is released and harnessed to do mechanical work. 36 Topological Relationships Between Compartments of the Secretory and Endocytic Pathways in a Eucaryotic Cell

Figure 12-5

Topologically equivalent spaces are shown in red. In principle, cycles of membrane budding and fusion permit the lumen of any of these organelles to communicate with any other and with the cell exterior by means of transport vesicles. Blue arrows indicate the extensive outbound and inbound vesicular traffic. Some organelles, most notably mitochondria and (in plant cells) plastids, do not take part in this communication and are isolated from the traffic between organelles shown here. 37 The Constitutive and Regulated Secretory Pathways

Figure 13-63 38 Multiple Models for the Mechanism of Nuclear Transport

(Fig.5.42 CELLS, 2006) (Fig.5.43 CELLS, 2006)

The contacts between Kaps and Nups via The interaction between FG repeats prevent most FG repeats are the key to understanding proteins from translocating through the NPC. how translocation through the nuclear Proteins that contain binding sites for FG repeats pore occurs. can disrupt those interactions and partition through the NPC. 39