Introduction and Goals
In this tutorial we will continue to discuss the endomembrane system, specifically the flow of material between the endomembrane system and the plasma membrane. You will read about vesicle-mediated transport of material across the plasma membrane, the transport of material from the Golgi complex to the plasma membrane, and the transport of plasma membrane proteins and extracellular material to the lysosome. You will review the functions of the endosome and the lysosome, and learn about the mechanisms that drive vesicle formation, vesicle targeting, and fusion of vesicles. Finally, the unique role of the plant vacuole will be described.
By the end of this tutorial you should know:
- The mechanisms of constitutive and regulated secretion
- The mechanisms of phagocytosis, bulk-phase endocytosis, and receptor-mediated endocytosis
- The endosomal pathway and the sorting of endocytosed material
- The role of coat proteins in vesicle formation, and the selection of proteins included in the vesicle
- The role of SNARE proteins in targeting, and the fusion of vesicles to the correct membrane
- The function of the lysosome in intracellular digestion
- The diverse functions of the plant vacuole in intracellular digestion, storage, regulation of turgor pressure, and cell size
Introduction to Exocytosis and Endocytosis
Exocytosis and endocytosis are vesicle-mediated transport of materials across the plasma membrane. Exocytosis is the fusion of secretory vesicles with the plasma membrane to release their contents into the extracellular environment. Endocytosis is the import of extracellular molecules by invagination of the plasma membrane to create a vesicle.
Constitutive and regulated secretion
The most common form of exocytosis is the secretion of proteins. Secretory vesicles, derived from the trans face of the Golgi complex, fuse with the plasma membrane (see Figure 1). As a result of fusion, the contents of the vesicles are discharged into the extracellular environment. In addition, the vesicles' lipid bilayers are integrated into the plasma membrane, increasing its surface area. Two distinct types of secretory vesicles undergo exocytosis: constitutive secretory vesicles and regulated secretory vesicles. Both are derived from the Golgi complex, but they contain distinct types of secreted proteins. Constitutive secretory vesiclescarry proteins that are continuously secreted by the cell. These vesicles are pinched off from the trans face of the Golgi complex, travel along microtubule tracks, and immediately fuse with the plasma membrane, thus releasing their contents. Many unrelated proteins can be packaged into a single constitutive secretory vesicle.
Regulated secretory vesicles are also derived from the trans face of the Golgi complex, however, they do not fuse with the plasma membrane until the cell is signaled to secrete. These secretory vesicles often contain protein hormones, digestive enzymes and neuro-peptides, depending on the cell type. These secretory vesicles are stored in the cytoplasm, where they aggregate and concentrate their contents. For some of these packaged proteins, proteolytic cleavage triggers their activation. Fusion of a secretory vesicle with the plasma membrane, and subsequent secretion of its stored proteins, occurs in response to a signal often associated with an increase in intracellular calcium. The peptide hormone insulin, which is secreted by cells in the pancreas in response to high levels of glucose in the bloodstream, is an example of regulated secretion. The secretion of insulin promotes the uptake of glucose from the bloodstream and the synthesis of glycogen.
Many cell types exhibit polarized secretion. Here, the secretion of specific proteins is restricted to one surface of the plasma membrane. For example, the epithelial cells lining your digestive tract are active in the secretion of digestive enzymes; however, secretion is limited to one surface of the cell, the apicalsurface, which is the portion of the plasma membrane facing the lumen of the intestine. Proteins destined for the same region of the plasma membrane will often be associated with lipid rafts and packaged into the same secretory vesicles.
Phagocytosis and Pinocytosis
Endocytosis occurs through invagination, or pinching off, of the plasma membrane to create an endocytic vesicle. In this process, extracellular materials and cell surface molecules are internalized. There are two types of endocytosis: phagocytosis and pinocytosis. Phagocytosisis the internalization of large particles, usually aggregates of macromolecules or microorganisms, from the extracellular environment. Few eukaryotic cells undergo phagocytosis, however, specialized white blood cells termed macrophages do so as a means of defense against infection. Macrophages recognize and destroy bacteria and cellular debris in the bloodstream. The mechanism is illustrated in Figure 2. The plasma membrane of a macrophage engulfs the bacterium and encapsulates it, creating a specialized vesicle termed a phagosome. This vesicle will fuse with a late endosome or lysosome, which will finally digest the internalized bacterium.
Pinocytosis, the internalization of extracellular fluid, is more common in eukaryotic cells. There are two types of pinocytosis: bulk-phase endocytosis and receptor-mediated endocytosis. The term pinocytosis has become outdated, and these processes are generally referred to as endocytosis. Bulk-phase endocytosis is the nonspecific internalization of extracellular fluid. In some single-cell organisms this process is used to ingest nutrients. Its function is not clear in cells of multicellular organisms, however, on average, 2% of the plasma membrane is internalized per minute. During bulk-phase endocytosis, a small portion of the plasma membrane folds in and pinches off, forming a small endocytic vesicle (see Figure 3). Many, but not all, of these endocytic vesicles arise at a specialized portion of the plasma membrane called a clathrin-coated pit. Clathrin-coated pits are regions of the membrane that have a network of the protein clathrinjuxtaposed to the cytoplasmic surface. Clathrin is one of several types of coat complex proteins (to be discussed later in this tutorial) that drive the formation of vesicles. The endocytic vesicles usually fuse with early endosomes, and membrane lipids and proteins are often recycled back to the plasma membrane.
Receptor-Mediated Endocytosis, Transcytosis, and the Endosomal Pathway
Receptor-mediated endocytosis is a distinct type of endocytosis in animal cells. This mechanism internalizes specific extracellular macromolecules through the binding to and internalization of cell surface receptors. One of the best-studied examples of receptor-mediated endocytosis is the uptake of cholesterol from the extracellular fluid. This is depicted in Figure 4. Most cholesterol in the bloodstream is associated with proteins, forming a complex termed low-density lipoproteins (LDLs). Cells take up cholesterol by the binding of LDL particles to LDL receptors, which are associated with clathrin-coated pits. Substances such as LDL particles that bind to a specific receptor molecule are termed ligands. Ligand-bound receptors accumulate at the coated pits, and then are internalized via endocytosis. The endocytic vesicle loses its clathrin coat and delivers its contents to an endosome. Here, the receptor and ligand dissociate; the receptor is recycled back to the plasma membrane and the LDL particle is transported to the lysosome. In the lysosome, the LDL particle is digested, thus releasing free cholesterol that can be used by the cell.
Transcytosis is the movement of receptor-bound macromolecules through the cell, employing both endocytosis and exocytosis. This occurs most commonly in polarized cells (e.g. epithelial cells). The best-studied example is the absorption and transport of antibodies across the epithelial lining of the gut. A newborn rat absorbs antibodies from its mother's milk. The antibody is bound to specific receptors on the apical surface of an intestinal cell (facing the lumen of the gut), and it is internalized via receptor-mediated endocytosis and transported to an early endosome. From there, the receptor-bound antibody moves to a recycling endosome and is delivered to the opposite surface of the cell, the basolateral surface. The receptor and antibody dissociate and the antibody can now enter the newborn rat's bloodstream.
The endosomal pathway
The endosomal pathway sorts and delivers endocytosed material to various compartments of the cell. Some material is destined for degradation in the lysosome, and some will be recyled back to the plasma membrane. As a result of endocytosis, an endocytic vesicle is formed, and it will fuse with an early endosome. This, in turn, will mature into a late endosome, and finally into a lysosome (Figure 5). The early endosome is where sorting of internalized material and membrane is thought to occur. A region of the early endosome will pinch off, creating a recycling endosome containing membrane and receptor molecules to be recycled back to the plasma membrane. The remainder of the early endosome will mature into the late endosome. There are two routes for the delivery of the material in the late endosome to the lysosome. The late endosome can mature into the lysosome by fusion with transport vesicles that have budded from the Golgi complex (which contain the newly synthesized and modified lysosomal hydrolases; see Tutorial Intracellular Components- ER and Golgi Complex). Alternatively, the endosome can fuse with a preexisting lysosome.
Vesicle Formation, Targeting and Fusion
The movement of material by endocytosis and exocytosis is mediated by the formation of vesicles. In addition, you should recall that movement of material between the ER and Golgi complex is also mediated by vesicle transport. In this section we will consider the molecular mechanisms that regulate the formation of vesicles, the inclusion of proteins into distinct vesicles, and the targeting and fusion of vesicles to the correct membrane. These processes are mediated, in part, by specific proteins associated with the vesicle membrane.
Protein coats and vesicle formation
Most vesicles bud off from a portion of membrane that is covered with a protein lattice associated with the cytoplasmic surface of the membrane that mediates vesicle budding. These protein complexes serve two purposes: they mediate the curving and budding of the membrane to form a coated vesicle; and they select the material to be included in the vesicle, referred to as the cargo. These proteins, initially associated with the budding membrane, will eventually completely cover the vesicle, which is termed a coated vesicle. These proteins will also directly, or indirectly, interact with the proteins to be included in the vesicle. As you will learn, specific proteins mediate different types of vesicles.
You have already been introduced to one of these coat proteins, clathrin, and its role in vesicle formation during endocytosis. In addition to endocytic vesicles, clathrin is associated with the coated vesicles that move material between the trans face of the Golgi complex and the endosomes. Prior to fusing with an endosome, a clathrin-coated vesicle will shed its protein coat. Another type of protein complex that is associated with vesicles is COPII (coat complex II), which is located on vesicles that move material from the ER to the Golgi complex. The third type of protein coat is formed by COPI (coat complex I), which is found on vesicles that move in a retrograde fashion through the Golgi complex, and from the Golgi complex to the ER. One of the COPI protein subunits binds to the KDEL receptor in the Golgi complex that mediates the retrieval of soluble ER proteins from the Golgi complex to the ER (see previous tutorial). In the absence of this COPI subunit, newly synthesized ER proteins are not retrieved from the Golgi complex.
Although different, clathrin, COPI and COPII share some features. The mechanism of driving vesicle formation is basically the same for all three proteins complexes, as is illustrated in Figure 6. All polymerize along the cytoplasmic surface of a membrane. These proteins, or associated adapter proteins, bind to membrane cargo proteins and membrane receptors that bind soluble cargo proteins, thereby ensuring their inclusion in the newly formed vesicle. The actual budding of membranes is directed by small GTP-binding proteins.
SNARE Proteins, and Vesicle Targeting and Fusion
Once a coated vesicle is formed, it will travel through the cell along microtubules until it reaches the membrane that it will fuse with, its target membrane. Prior to docking with the target membrane, the coated vesicle will lose its coat and expose a class of integral membrane proteins termed SNAREs. SNAREs are SNAP (described below) receptors located on the membrane surface that mediates selective targeting and fusion of vesicles. These proteins and the mechanism of their action are illustrated in Figure 7. There are two categories of SNARES: v-SNAREs, located on the vesicle membrane, and t-SNAREs, located on the target membrane. Each distinct SNARE is associated with a different membrane component of the cell. The complementary v-SNARE and t-SNARE of apposing membranes interact with each other to form a coiled coil, bringing the two membranes in close contact and facilitating fusion.
SNAREs are not the only proteins needed for correct vesicle targeting and fusion. Mutation analyses have shown that cells missing a particular SNARE will still undergo relatively normal vesicle targeting and fusion, indicating that other proteins probably are important. Distinct Rab proteins, a class of GTP-binding proteins, are distributed on each membrane compartment of the cell. Rabs appear to function in the selective tethering of vesicles to target membranes prior to docking and fusion mediated by SNAREs. In addition, other proteins termed SNAPs (soluble NSF (see below) attachment proteins) are required to stabilize the pre-fusion complex. NSFs (N-ethyl maleimide sensitive factors) are required to dissociate the SNARE complexes in order to free the proteins for further interactions and membrane fusion. The molecular details of this pathway are still being determined, and it is likely that the role of additional proteins will emerge.
The Lysosome is the Site of Intracellular Digestion
The lysosome is responsible for degradation of macromolecules in animal cells. The mature lysosome is filled with many acid hydrolases, hydrolytic enzymes that are active at low pHs. These include nucleases (enzymes that degrade nucleic acids), proteases (enzymes that degrade proteins), glycosidases (enzymes that degrade oligosaccharides and polysaccharides), and lipases (enzymes that degrade lipids). The lysosome is also distinct because it maintains a low interior pH. There are several distinct pathways that deliver material to the lysosome, one of which was eluded to already in our discussion of the endosomal pathway. An endocytic vesicle fuses with an early endosome; this, in turn, will mature into a late endosome, and finally, into a lysosome. The material delivered to the lysosome will be degraded, and in some cases, the breakdown products such as amino acids, nucleotides and sugars will be transported to the cytoplasm and recycled by the cell. In a macrophage undergoing phagocytosis, bacteria are encapsulated in a phagosome that will fuse with a late endosome or lysosome, which will finally digest the internalized bacteria. Another key role of the lysosome is autophagy, which is the degradation of unwanted organelles. The organelle is surrounded by endosomal membrane and is targeted for degradation in a fashion similar to a phagosome. Finally, in some cell types the lysosome undergoes exocytosis, releasing its contents into the environment.
The plant vacuole has many functions, including intracellular digestion
Most plant cells contain large fluid-filled vesicles called vacuoles. Vacuoles can occupy from 30% - 90% of a cell's volume. The vacuole is related to the lysosome, containing some of the same hydrolytic enzymes and maintaining a low interior pH; however, the vacuole is not as specialized as the lysosome. Vacuoles function as storage vesicles for nutrients and waste products. Both cell size and turgor pressure(the osmotic pressure against the cell wall) are regulated by vacuoles. The vacuoles maintain a much higher ionic concentration than the cytoplasm, and as a result water enters by osmosis. The osmotic pressure in vacuoles maintains the cells' turgor pressure, which keeps plant tissues from wilting and provides growing cells with the force necessary to expand.
The processes of exocytosis and endocytosis are vesicle-mediated mechanisms for transporting materials across the plasma membrane. The most common form of exocytosis is secretion. Constitutive secretion comprises the bulk flow of vesicles from the Golgi complex that fuse with the plasma membrane. In regulated secretion, vesicles bud from the Golgi complex and are stored in the cytoplasm until a signal is received to trigger exocytosis. Polarized secretion is restricted to a particular region of the plasma membrane, usually the apical surface of the cell. Phagocytosis is one form of endocytosis. It is the internalization of a relatively large particle, such as a macrophage engulfing a bacterium. Bulk-phase endocytosis is the nonspecific internalization of extracellular fluids and plasma membranes. Receptor-mediated endocytosis is the specific internalization of molecules that are bound to cell surface receptors, such as cholesterol bound to the LDL receptor. Here, an endocytic vesicle is created and it will fuse with an early endosome, the contents of which are sorted for further transport. Some material, such as membrane receptors, will be recycled back to the plasma membrane, while other components will be retained as the vesicle matures into a late endosome, and finally, into a lysosome (where it will be degraded). Transcytosis is a process by which material is internalized in one region of a cell (e.g. the apical surface) and delivered via recycling endosomes to another portion of the plasma membrane.
Vesicle formation is driven by specific protein complexes that act in conjunction with GTP-binding proteins. These proteins provide a scaffold that promotes the bending and pinching off of the membrane during vesicle formation. In addition, they selectively bind proteins to be included in the vesicle. These proteins are clathrin, COPI and COPII. They mediate vesicle formation in a similar fashion, but are associated with distinct vesicles. Correct vesicle docking and fusion with its target membrane are regulated, in part, by complementary v- and t-SNARES, as well as other proteins such as Rab, SNAPs and NSF.
The lysosome is responsible for intracellular degradation of many macromolecules. It is characterized by the acid hydrolases and a low interior pH. Material is delivered to the lysosome via the endosomal pathway. In addition to degradation of endocytosed material, the lysosome can break down organelles in a process referred to as autophagy. Finally, plants have a unique organelle, the vacuole, which has many diverse functions, including degradation of intracellular material (similar to the lysosome). Vacuoles also serve as storage organelles and regulate turgor pressure and cell size.