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Moving Components Through the Cell
C H A P T E R
17 Moving Components Through the Cell: Membrane Trafficking O U T L I N E 1. Introduction
369
2. Endo-Membrane Flow (Secretion)
370
3. Receptor-Mediated Endocytosis
372
4. The Golgi
374
5. Intracellular Lipid Transport 5.1 Lipid Vesicles 5.2 Lipid Transfer Proteins
375 376 376
5.3 Lipid Lateral Diffusion Through Membranes 5.4 Free Diffusion Through the Cytosol 5.5 Membrane-to-Membrane Contact 5.6 Lipid Flip-Flop
377 378 378 378
6. Summary
378
References
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1. INTRODUCTION If one were able to sit inside a cell and look around, one would be witness to a bewildering array of every type of life component (proteins, lipids, nucleic acids, membrane vesicles, etc.) zipping around in all directions, heading to specific cellular locations. It would first appear that everything is in total chaos, but on closer examination, well-designed patterns would emerge [1,2]. Movement of membrane-derived vesicles, referred to as “membrane trafficking,” is of particular interest as it forms the heart of the endo-membrane concept. Three major patterns will be discussed here: endo-membrane flow (for secretion), receptormediated endocytosis (RME), and intracellular carrier proteins.
An Introduction to Biological Membranes http://dx.doi.org/10.1016/B978-0-444-63772-7.00017-8
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© 2016 Elsevier B.V. All rights reserved.
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2. ENDO-MEMBRANE FLOW (SECRETION) As discussed in Chapter 16, there are two basic types of protein synthesis: one made on cytosolic ribosomes for water-soluble cytosolic proteins and the other made on identical ribosomes attached to the outer (cytosolic) surface of the endoplasmic reticulum (ER) for generating transmembrane proteins, proteins to be delivered to the inside of cellular organelles and for proteins to be secreted from the cell. The process of movement through the cell from the nucleus to the plasma membrane with stops at different intracellular organelles is known as “endo-membrane flow” (Fig. 17.1). The first proposal that intracellular membranes form a single interconnected system (the endo-membrane concept) was by Morre and Mollenhauer in 1974 [3]. Here, steps will be followed for secretion of proteins initially synthesized in the lumen of the rough ER (RER) [4]. Steps involved in protein synthesis on an ER-bound ribosome are discussed in Chapter 16. Once inside the RER lumen, the newly synthesized protein begins its modification process [5]. The first step involves chaperone proteins that help fold the newly synthesized protein. If the initial folding is incorrect, a second attempt is made. If this too fails, the misfolded protein is
FIGURE 17.1 Endo-membrane flow from the nucleus to the plasma membrane. Copyright McGraw Hill. http:// www.studyblue.com/notes/note/n/test-2-ch-12-/deck/5782652.
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exported to the cytosol and labeled for destruction (see ubiquitin in Chapter 16). For the correctly folded protein, carbohydrates are then added to convert the new protein into a glycoprotein. The sugars are required to help further correctly fold the protein, targeting the glycoprotein to a final destination and for proper functioning of the protein once secreted. The modified proteins are encapsulated into transport vesicles that bud off from the ER. These vesicles are covered by a coating protein COPII (coat protein complex II) and are moved to the cis face of the Golgi (the Golgi is discussed in detail later in Chapter 17, Section 4). In passing through the Golgi, the protein is further modified before being dispatched from the trans face of the Golgi in a different type of uncoated transport vesicle. These vesicles are carried by the cytoskeleton to the plasma membrane for fusion and release of its contents to the extra-cellular solution (secretion). The transport (secretory) vesicles have surface components that recognize, and bind to receptors on the cytoplasmic side of the plasma membrane [6]. The process of secreting a protein follows the same basic steps that cells use to excrete waste and other large molecules from the cytoplasm to the cell exterior [7] and therefore is the opposite of endocytosis (discussed in Section 3). Fusion of the transport vesicles to the plasma membrane not only releases their aqueous sequestered contents to the outside but at the same time also adds vesicular membrane hydrophobic components (mostly lipids and proteins) to the plasma membrane. The process is depicted in Fig. 17.2 [8]. Steady state composition of the plasma membrane results from a balance between endocytosis and
FIGURE 17.2 Mechanism of formation of transport (secretory) vesicles, their fusion to the plasma membrane, and release of sequestered material. From The Rye Laboratory, Department of Biochemistry and Biophysics. Texas A&M University. http://ryeserv1.tamu.edu/secretory-vesicle-formation.html.
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exocytosis. The resultant process of plasma membrane recycling is amazingly fast. For example, pancreatic secretory cells recycle an amount of membrane equal to the whole surface of the cell in about 90 min. Even faster are macrophages that can recycle contents of the plasma membrane in only 30 min. The transport vesicles must first dock with the plasma membrane, a process that keeps the two membranes separated by less than 5e10 nm. During docking, complex molecular rearrangements occur to prepare the membranes for fusion where separation becomes less than 1.5 nm. The process of vesicle fusion and release of aqueous compartment components is driven by SNARE proteins (see Chapter 10) [9]. Joining the vesicle SNARE (v-SNARE) to the plasma membrane SNARE (t-SNARE) is checked and locked by the regulatory GTPbinding protein Rab. Therefore, the process of exocytosis results in: • The surface of the plasma membrane increasing by the size of the fused vesicular membrane. This is particularly important if the cell is growing. • The material sequestered inside the vesicle is released to the cell exterior. Included in the vesicle contents may be waste products, intracellular toxins, and signaling molecules like hormones and neurotransmitters. • Proteins imbedded in the vesicular membrane become part of the plasma membrane. This makes correct protein orientation in the vesicular membrane absolutely essential. The side of the protein facing the inside of the vesicle before fusion faces the outside of the plasma membrane after fusion. • Some variations in the standard vesicle fusion process exist. Presynaptic vesicles carrying neurotransmitters do not fuse immediately but instead must await a fusion initiation signal (Chapter 18). A special type of exocytosis called “kiss-and-run” occurs in synapses [10]. The vesicles only make very brief contact with the plasma membrane whereupon they release their contents (neurotransmitters) to the outside (synaptic gap) and immediately return empty to the neuron cytoplasm. Since true fusion does not occur, the vesicle membrane is not incorporated into the plasma membrane. For their work on the structure and function of cellular vesicles, James Rothman, Randy Schekman, and Thomas Sudhof shared the 2013 Nobel Prize in Physiology or Medicine.
3. RECEPTOR-MEDIATED ENDOCYTOSIS RME [11,12] is also known as clathrin-dependent endocytosis because of involvement of the membrane-associated protein clathrin in forming membrane vesicles that become internalized into the cell. Clathrin plays a major role in formation of clathrin-coated pits and coated vesicles. Since clathrin was first isolated and named by Barbara Pearse in 1975 [13], it has become clear that clathrin and other coat proteins play essential roles in cell biology. Clathrin is an essential component in building small vesicles for uptake (endocytosis) and export (exocytosis) of many molecules. While the methods of membrane transport, discussed in Chapter 19, involved small solutes, RME is the primary mechanism for the specific internalization of most macromolecules by eukaryotic cells.
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3. RECEPTOR-MEDIATED ENDOCYTOSIS
Clathrin-dependent endocytosis receptor
formation of clathrin-coated vesicles PM
AP-2 clathrin uncoating AP-2 complex α-adaptin β2-adaptin μ2-chain
fusion
σ2-chain early endosomes
FIGURE 17.3 Receptor mediated endocytosis (also known as clathrin-dependent endocytosis). Reference Grant BD, Sato M. http://www.wormbook.org/chapters/www_intracellulartrafficking/intracellulartrafficking.html; 2006.
RME begins with an external ligand binding to a specific receptor that spans the plasma membrane (Fig. 17.3, [14,15]). Examples of these ligands include hormones, growth factors, enzymes, serum proteins, low-density lipoprotein (LDL) (with attached cholesterol), transferrin (with attached iron), antibodies, some viruses, and even bacterial toxins. After receptor binding, the complex diffuses laterally in the plasma membrane until it encounters a specialized patch of membrane called a coated pit. The receptoreligand complexes accumulate in these patches as do other proteins including clathrin, adaptor protein, and dynamin. Since coated pits occupy about 20% of the plasma membrane surface area, they are not minor membrane features. The collection of these proteins starts to curve the adjacent section of the membrane that eventually pinches off to form an internalized coated vesicle. Clathrin and dynamin then recycle back to the plasma membrane, leaving an uncoated vesicle that is free to fuse with an early endosome. After the early endosomes mature into late endosomes, they then go to the lysosome for digestion. RME is a very fast process. Invagination and vesicle formation take about 1 min. One single cultured fibloblast cell can produce 2500 coated pits per minute. One example of RME has received a great deal of attention because of its essential role in human health, namely maintaining the proper level of cholesterol in the body. Malfunctions in the RME process for uptake of cholesterol-carrying LDL (see Chapter 14) leads to hypercholesterolemia and cardiovascular disease [11,16]. RME and its role in cholesterol metabolism was discovered by Michael Brown and Joseph Goldstein of The University of Texas Health Science Center in Dallas (now the UT Southwestern Medical Center), who received the 1985 Nobel Prize in Physiology and Medicine for their iconic work.
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4. THE GOLGI Endo-membrane flow, RME, and many other essential cell functions use the Golgi as a central component [17]. The Golgi is often referred to as the “Post Office of the Cell” because its major functions involve packaging and labeling items to be shipped to various destinations around the cell. The Golgi is an intracellular organelle whose large size facilitated its early discovery and morphological description by Italian physiologist Camillo Golgi (Fig. 17.4) in 1897 (see also Chapter 18). After Golgi announced his discovery at a meeting of the Medical Society of Pavia on April 19, 1898, the organelle was named after him. To this day, the Golgi is the only organelle named after a scientist. Discovery of the organelle was facilitated by Golgi’s development of silver as a stain for nerve cells. For his work, Camillo Golgi was awarded the 1906 Nobel Prize in Physiology or Medicine, one of the earliest Nobel Prizes. (In 1901, the first Nobel Prize in Physiology or Medicine went to the German physiologist Emil Adolf van Behring for his work on serum therapy and development of a vaccine against diphtheria.) Golgi’s Italian birth town is now named Corteno Golgi in his honor. The number and size of Golgi vary from cell to cell. Golgi tend to be larger and more numerous in cells that have a large secretion function. Morphologically, the Golgi appears to be composed of membrane-bound stacked structures called cisternae [18]. A “typical” Golgi is depicted in Fig. 17.5. A single mammalian cell usually contains between 40 and 100 Golgi stacks, each of which is composed of 4 to 8 cisternae. Each cisternae is a flat, membrane-enclosed disc that houses the enzymes that conduct the Golgi’s business. The central spaces of the stacked cisternae are contiguous, allowing for processing of cargo as it passes through the Golgi [17]. Each cisternae is composed of four functional regions or networks referred to as the cis-Golgi, medial-Golgi, endo-Golgi, and trans-Golgi. Transport vesicles originating from the ER fuse with the cis-Golgi, releasing their cargo into the lumen of the cisternae. The cargo is modified as it passes through the Golgi, eventually reaching the trans-Golgi, where it is sorted, packaged, and sent to its final destination. Each region of the Golgi contains characteristic enzymes that selectively modify the cargo.
FIGURE 17.4
Camillo Golgi, 1843e1926. http://www.vetmed.vt.edu/education/curriculum/vm8304/lab_companion/ histo-path/vm8054/labs/Lab3/Notes/golginot.htm.
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FIGURE 17.5
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Diagram of a “typical” Golgi. http://www.glogster.com/johnv4297/john-v-s-glog/g-6ltb1bblffg5cerho
4nuna0.
As the cargo proteins pass through the connected Golgi lumen, many modifications are made to ready the proteins for sorting, packaging, and eventual shipment to their appropriate destinations. Most modifications involve glycosylations, primarily to existing sugar chains, although phosphorylations and sulfations are also common. Some of the newly attached sugars may function as a type of signal, directing the protein to the appropriate transport vesicle forming in the trans-Golgi. One modification that has been known for decades involves attaching mannose-6-phosphate, targeting the protein to the lysosome. Sulfation is generally performed by the enzyme sulfotransferase in the trans-Golgi. Added sulfates give the cargo protein a net negative charge that helps in sorting. Cargo proteins destined for secretion or a particular organelle, depart the trans-Golgi in destination-specific transport vesicles and deposit their cargo at the target membrane via fusion.
5. INTRACELLULAR LIPID TRANSPORT While the trafficking of membrane proteins through a cell can usually be followed precisely, the trafficking of membrane lipids is far more troublesome. To begin with for every protein there are 50 or more lipids found in its immediate environment, and these lipids are composed of many different molecular species (see Chapter 10). So, how are membrane lipids transported throughout a cell? While details remain elusive, several overlapping
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mechanisms are possible. Two of these mechanisms, serum albumin and lipoproteins, were discussed in Chapter 14. Other mechanisms include: 1. 2. 3. 4. 5. 6.
Vesicle transport Lipid transfer proteins (LTPs) Lipid lateral diffusion through membranes Free diffusion through the cytosol Membrane to membrane contact Lipid flip-flop
5.1 Lipid Vesicles A living cell is chock-full of lipid vesicles. In fact, there are so many vesicles that for many years cell biologists thought the vesicles were merely artifacts of electron microscopy procedures. A prime example is the secretory vesicles discussed earlier that are involved in membrane trafficking, endo-membrane flow, secretion, and RME. When one of these vesicles fuses with a cell membrane, an enormous number of vesicular membrane lipids are mixed with the cell membrane lipids, increasing the membrane size and altering the membrane lipid composition. This mechanism of lipid transfer likely moves mere lipids than does the other mechanisms. However, vesicle fusion does not have the ability to accurately control the type and amount of lipid transferred.
5.2 Lipid Transfer Proteins LTPs are essential for the movement of lipids both intercellularly and intracellularly and help develop and maintain lipid compositions characteristic of organelles and membrane domains. LTPs were discovered by D.B. Zilversmidt in 1968 as soluble factors that accelerated the transfer of lipids between membranes [19]. We have already encountered an early example of an LTP called a phospholipid exchange protein (PLEP, see Chapter 9). PLEPs have been extensively used to measure transmembrane lipid asymmetry and flip-flop. Since their initial discovery, LTPs have been found in all eukaryotes and bacteria. They come in a wide variety of types, accounting for their ability to transfer many structurally diverse membrane lipids [20,21]. In general, LTPs mediate monomeric lipid exchange in which a single lipid molecule is transported through the aqueous phase sequestered in a hydrophobic pocket of the LTP. Most LTPs are low molecular weight structures that are dominated by b-sheet motifs (Fig. 17.6). A “lid” covers the opening to the hydrophobic pocket and acts as the gate for lipid uptake and release. Thus, LTPs exist in two distinct conformations, a closed conformation that transports the lipid through the aqueous phase and an open conformation where the LTP picks up its lipid cargo at the outer leaflet of a donor membrane and releases it to the outer leaflet of an acceptor membrane. Sterol carrier proteins (also known as nonspecific lipid transfer proteins [NSLTPs]) are a family of LTPs that can transfer a variety of lipids from one intracellular membrane to another [22,23]. They are referred to as “nonspecific” since they have been reported to carry sterols, glycolipids, all common phospholipids, and gangliosides. The sterol carrier proteins
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FIGURE 17.6 Structure of the lipid transfer protein, the sterol carrier protein-2 (SCP-2). http://upload.wikimedia.org/ wikipedia/commons/c/cc/Protein_SCP2_PDB_1c44.png.
are actually two separate proteins: sterol carrier protein X (SCPx, 46 kDa) and the much smaller (13 kDa) sterol carrier protein-2 (SCP-2 is depicted in Fig. 17.6). The SCPs have been suggested to play a role in Zellweger syndrome, a peroxisome disorder. Many other examples of LTPs exist for specific types of lipids: ceramide-transfer protein (CERT), sphingolipid-transfer proteins (CERT and FAPP2), phosphatidylcholine-transfer protein (PCTP), phosphatidylinositol-transfer protein (PITP), retinoid binding proteins (RBPs), and a-tocopherol transfer protein. In mammals, PCTPs, PITPs, and NSLTPs are the three major classes of phospholipid transfer proteins.
5.3 Lipid Lateral Diffusion Through Membranes Although vesicle transfer and LTPs are the most appreciated mechanisms for lipid movement, other significant processes exist. One of the most important of these is lateral diffusion through the membrane plane. Lateral diffusion is responsible for lipid movement through endo-membrane flow. As discussed in Chapter 9, lipids can diffuse rapidly, moving completely around a liver cell in about 1 min. After transport vesicles fuse with a membrane, the newly added lipids rapidly spread out from the site of fusion via lateral diffusion. Lipid lateral diffusion rates are affected by many membrane properties, including lipid bilayer phase, type of lipid micro-domain, lipid heterogeneity, membrane lateral pressure, lipide lipid affinities, lipideprotein affinities, membrane protein crowding, lipid interdigitation, and lipidecytoskeleton interactions (Chapter 10). Lipid vesicle formation is also affected by the membrane lipid content, particularly the acyl chain double bond content, and levels of PS (phosphatidylserine), PA (phosphatidic acid), and DAG (diacylglycerol).
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5.4 Free Diffusion Through the Cytosol Initially, it would seem feasible that membrane lipids could simply be ejected from the bilayer and diffuse across the aqueous compartment to a distant membrane. While theoretically possible, the extremely low solubility of membrane lipids in water would preclude this mechanism from being significant (see Chapter 13, Detergents).
5.5 Membrane-to-Membrane Contact Lipid transfer also occurs at points of contact between distant sites on a membrane.
5.6 Lipid Flip-Flop Finally, lipid transfer is not only associated with diffusion laterally through the membrane plane but also transversely across the membrane in a process known as flip-flop (see Chapter 9). Inherently, lipid flip-flop is a slow event that can be greatly accelerated by proteinelipid interfaces and by enzymes known as flipases, flopases, and scrambleases (see Chapter 9). Many examples of transfer of a specific lipid from one membrane to another for enzymatic modification have been reported [20]. For example, ER PS is transferred to the mitochondria, where it is decarboxylated to PE before being sent back to the ER. Surprisingly, this bidirectional, nonvesicular lipid transfer accounts for most PE synthesis in mammalian cells. Ceramide is also synthesized in the ER and is primarily transported by CERT to the Golgi, where it is converted into sphingomyelin (SM) by SM synthase. It is clear that in cells, membrane lipids are constantly on the move via a variety of mechanisms.
6. SUMMARY A characteristic of all living cells is the continuous movement of material from place to place within the cell. Translocation of membrane-associated constituents is referred to as “membrane trafficking” and forms the heart of the endo-membrane concept. Discussed here are methods commonly used in translocation: secretion (exocytosis), RME, and intracellular carrier proteins. A central component of the endo-membrane flow theory is the Golgi apparatus, a type of molecular processing center that takes in materials from various parts of the cell, modifies and repackages them, and sends them to new cellular locations. Both exocytosis (using SNARE fusion proteins) and endocytosis (through clathrin-coated pits) are largely responsible for the enormous number of intracellular vesicles. Intracellular vesicle fusion to membranes, and a large family of specific and nonspecific LTPs (lipid transfer proteins) are primarily responsible for the rapid movement of polar lipids from one cellular membrane to another. Chapter 18 will discuss involvement of membranes in the essential cell functions of: anesthetic action, G proteinecoupled reactions, membrane attack complex, nerve conduction, and electron transport/oxidative phosphorylation.
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References [1] Chaung AY, de Vries SC. Membrane trafficking: intracellular highways and country roads. Plant Physiol 2008;147(4):1451e3. [2] Fu’rthauer M, Gonza’lez-Gaita’n M. Tales of 1001 functions: the multiple roles of membrane trafficking in development. (Editorial). Traffic 2009;10:781e2. [3] Morré DJ, Mollenhauer HH. The endomembrane concept: a functional integration of endoplasmic reticulum and Golgi apparatus. In: Robards AW, editor. Dynamic aspects of plant infrastructure. New York: McGraw-Hill; 1974. p. 84e137. [4] Lodish H, Berk A, Zipursky SL, et al. Overview of the secretory pathway. Molecular cell Biology. Section 17.3. 4th ed. New York: W.H. Freeman; 2000. [5] Vitale A, Denecke J. The endoplasmic reticulumdgateway of the secretory pathway. Plant Cell 1999;11(4):615e28. [6] Anderson LL. Discovery of the ‘porosome’ the universal secretory machinery in cells. J Cell Mol Med 2006;10(1):126e31. [7] Li L, Chin L-S. The molecular machinery of synaptic vesicle exocytosis. Cell Mol Life Sci 2003;60:942e60. [8] Cell and Cell Structure IV. Membrane Transport Processes. Benjamin Cummings an imprint of Addison Wesley Longman. Cell and cell structure. IV. Membrane transport processes. 2001. http://www.highlands.edu/ academics/divisions/scipe/biology/faculty/harnden/2121/notes/cell.htm. [9] Sudhof TC, Rothman JE. Membrane fusion: grappling with SNARE and SM proteins. Science 2009;323:474e7. [10] Wightman RM, Haynes CL. Synaptic vesicles really do kiss and run. Nat Neurosci 2004;7:321e2. [11] Goldstein JL, Brown MS, Anderson RGW, Russell DW, Schneider W. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Ann Rev Cell Biol 1985;1:1e39. [12] Wileman T, Harding C, Stahl P. Receptor-mediated endocytosis. Biochem J 1985;232:1e14. [13] Pearse BM. Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles. Proc Natl Acad Sci U S A 1976;73:1255e9. [14] Grant BD, Sato M. http://www.wormbook.org/chapters/www_intracellulartrafficking/intracellular trafficking.html; 2006. [15] McMahon HT, Boucrot E. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 2011;12:517e33. [16] Brown MS, Goldstein JL. Receptor-mediated control of cholesterol metabolism. Science 1976;191:150e4. [17] Glick BS, Nakano A. Membrane traffic within the Golgi apparatus. Annu Rev Cell Dev Biol 2009;25:113e32. [18] Mollenhauer HH, Morre DJ. Structure of Golgi apparatus. Protoplasma 1994;180:14e28. [19] Wirtz KWA, Zilversmit DB. Exchange of phospholipids between liver mitochondria and microsomes in vitro. J Biol Chem 1968;243:3596e602. [20] Lev S. Non-vesicular lipid transport by lipid-transfer proteins and beyond. Mol Cell Biol 2010;11:739e50 [01.10.10]. [21] Wirtz KWA. Phospholipid transfer proteins. Ann Rev Biochem 1991;60:73e99. [22] Stolowich NJ, Petrescu AD, Huang H, Martin GG, Scott AI, Schroeder F. Sterol carrier protein-2: structure reveals function. Cell Mol Life Sci 2002;59:193e212. [23] Schroedr F, Huang H, McIntosh AL, Atshaves BP, Martin GG, Kier AB. Caveolin, sterol carrier protein-2, membrane cholesterol-rich microdomains and intracellular cholesterol trafficking. Subcell Biochem 2010;51:279e318.
II. MEMBRANE BIOLOGICAL FUNCTIONS
17 Moving Components Through the Cell: Membrane Trafficking O U T L I N E 1. Introduction
369
2. Endo-Membrane Flow (Secretion)
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3. Receptor-Mediated Endocytosis
372
4. The Golgi
374
5. Intracellular Lipid Transport 5.1 Lipid Vesicles 5.2 Lipid Transfer Proteins
375 376 376
5.3 Lipid Lateral Diffusion Through Membranes 5.4 Free Diffusion Through the Cytosol 5.5 Membrane-to-Membrane Contact 5.6 Lipid Flip-Flop
377 378 378 378
6. Summary
378
References
379
1. INTRODUCTION If one were able to sit inside a cell and look around, one would be witness to a bewildering array of every type of life component (proteins, lipids, nucleic acids, membrane vesicles, etc.) zipping around in all directions, heading to specific cellular locations. It would first appear that everything is in total chaos, but on closer examination, well-designed patterns would emerge [1,2]. Movement of membrane-derived vesicles, referred to as “membrane trafficking,” is of particular interest as it forms the heart of the endo-membrane concept. Three major patterns will be discussed here: endo-membrane flow (for secretion), receptormediated endocytosis (RME), and intracellular carrier proteins.
An Introduction to Biological Membranes http://dx.doi.org/10.1016/B978-0-444-63772-7.00017-8
369
© 2016 Elsevier B.V. All rights reserved.
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2. ENDO-MEMBRANE FLOW (SECRETION) As discussed in Chapter 16, there are two basic types of protein synthesis: one made on cytosolic ribosomes for water-soluble cytosolic proteins and the other made on identical ribosomes attached to the outer (cytosolic) surface of the endoplasmic reticulum (ER) for generating transmembrane proteins, proteins to be delivered to the inside of cellular organelles and for proteins to be secreted from the cell. The process of movement through the cell from the nucleus to the plasma membrane with stops at different intracellular organelles is known as “endo-membrane flow” (Fig. 17.1). The first proposal that intracellular membranes form a single interconnected system (the endo-membrane concept) was by Morre and Mollenhauer in 1974 [3]. Here, steps will be followed for secretion of proteins initially synthesized in the lumen of the rough ER (RER) [4]. Steps involved in protein synthesis on an ER-bound ribosome are discussed in Chapter 16. Once inside the RER lumen, the newly synthesized protein begins its modification process [5]. The first step involves chaperone proteins that help fold the newly synthesized protein. If the initial folding is incorrect, a second attempt is made. If this too fails, the misfolded protein is
FIGURE 17.1 Endo-membrane flow from the nucleus to the plasma membrane. Copyright McGraw Hill. http:// www.studyblue.com/notes/note/n/test-2-ch-12-/deck/5782652.
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exported to the cytosol and labeled for destruction (see ubiquitin in Chapter 16). For the correctly folded protein, carbohydrates are then added to convert the new protein into a glycoprotein. The sugars are required to help further correctly fold the protein, targeting the glycoprotein to a final destination and for proper functioning of the protein once secreted. The modified proteins are encapsulated into transport vesicles that bud off from the ER. These vesicles are covered by a coating protein COPII (coat protein complex II) and are moved to the cis face of the Golgi (the Golgi is discussed in detail later in Chapter 17, Section 4). In passing through the Golgi, the protein is further modified before being dispatched from the trans face of the Golgi in a different type of uncoated transport vesicle. These vesicles are carried by the cytoskeleton to the plasma membrane for fusion and release of its contents to the extra-cellular solution (secretion). The transport (secretory) vesicles have surface components that recognize, and bind to receptors on the cytoplasmic side of the plasma membrane [6]. The process of secreting a protein follows the same basic steps that cells use to excrete waste and other large molecules from the cytoplasm to the cell exterior [7] and therefore is the opposite of endocytosis (discussed in Section 3). Fusion of the transport vesicles to the plasma membrane not only releases their aqueous sequestered contents to the outside but at the same time also adds vesicular membrane hydrophobic components (mostly lipids and proteins) to the plasma membrane. The process is depicted in Fig. 17.2 [8]. Steady state composition of the plasma membrane results from a balance between endocytosis and
FIGURE 17.2 Mechanism of formation of transport (secretory) vesicles, their fusion to the plasma membrane, and release of sequestered material. From The Rye Laboratory, Department of Biochemistry and Biophysics. Texas A&M University. http://ryeserv1.tamu.edu/secretory-vesicle-formation.html.
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exocytosis. The resultant process of plasma membrane recycling is amazingly fast. For example, pancreatic secretory cells recycle an amount of membrane equal to the whole surface of the cell in about 90 min. Even faster are macrophages that can recycle contents of the plasma membrane in only 30 min. The transport vesicles must first dock with the plasma membrane, a process that keeps the two membranes separated by less than 5e10 nm. During docking, complex molecular rearrangements occur to prepare the membranes for fusion where separation becomes less than 1.5 nm. The process of vesicle fusion and release of aqueous compartment components is driven by SNARE proteins (see Chapter 10) [9]. Joining the vesicle SNARE (v-SNARE) to the plasma membrane SNARE (t-SNARE) is checked and locked by the regulatory GTPbinding protein Rab. Therefore, the process of exocytosis results in: • The surface of the plasma membrane increasing by the size of the fused vesicular membrane. This is particularly important if the cell is growing. • The material sequestered inside the vesicle is released to the cell exterior. Included in the vesicle contents may be waste products, intracellular toxins, and signaling molecules like hormones and neurotransmitters. • Proteins imbedded in the vesicular membrane become part of the plasma membrane. This makes correct protein orientation in the vesicular membrane absolutely essential. The side of the protein facing the inside of the vesicle before fusion faces the outside of the plasma membrane after fusion. • Some variations in the standard vesicle fusion process exist. Presynaptic vesicles carrying neurotransmitters do not fuse immediately but instead must await a fusion initiation signal (Chapter 18). A special type of exocytosis called “kiss-and-run” occurs in synapses [10]. The vesicles only make very brief contact with the plasma membrane whereupon they release their contents (neurotransmitters) to the outside (synaptic gap) and immediately return empty to the neuron cytoplasm. Since true fusion does not occur, the vesicle membrane is not incorporated into the plasma membrane. For their work on the structure and function of cellular vesicles, James Rothman, Randy Schekman, and Thomas Sudhof shared the 2013 Nobel Prize in Physiology or Medicine.
3. RECEPTOR-MEDIATED ENDOCYTOSIS RME [11,12] is also known as clathrin-dependent endocytosis because of involvement of the membrane-associated protein clathrin in forming membrane vesicles that become internalized into the cell. Clathrin plays a major role in formation of clathrin-coated pits and coated vesicles. Since clathrin was first isolated and named by Barbara Pearse in 1975 [13], it has become clear that clathrin and other coat proteins play essential roles in cell biology. Clathrin is an essential component in building small vesicles for uptake (endocytosis) and export (exocytosis) of many molecules. While the methods of membrane transport, discussed in Chapter 19, involved small solutes, RME is the primary mechanism for the specific internalization of most macromolecules by eukaryotic cells.
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3. RECEPTOR-MEDIATED ENDOCYTOSIS
Clathrin-dependent endocytosis receptor
formation of clathrin-coated vesicles PM
AP-2 clathrin uncoating AP-2 complex α-adaptin β2-adaptin μ2-chain
fusion
σ2-chain early endosomes
FIGURE 17.3 Receptor mediated endocytosis (also known as clathrin-dependent endocytosis). Reference Grant BD, Sato M. http://www.wormbook.org/chapters/www_intracellulartrafficking/intracellulartrafficking.html; 2006.
RME begins with an external ligand binding to a specific receptor that spans the plasma membrane (Fig. 17.3, [14,15]). Examples of these ligands include hormones, growth factors, enzymes, serum proteins, low-density lipoprotein (LDL) (with attached cholesterol), transferrin (with attached iron), antibodies, some viruses, and even bacterial toxins. After receptor binding, the complex diffuses laterally in the plasma membrane until it encounters a specialized patch of membrane called a coated pit. The receptoreligand complexes accumulate in these patches as do other proteins including clathrin, adaptor protein, and dynamin. Since coated pits occupy about 20% of the plasma membrane surface area, they are not minor membrane features. The collection of these proteins starts to curve the adjacent section of the membrane that eventually pinches off to form an internalized coated vesicle. Clathrin and dynamin then recycle back to the plasma membrane, leaving an uncoated vesicle that is free to fuse with an early endosome. After the early endosomes mature into late endosomes, they then go to the lysosome for digestion. RME is a very fast process. Invagination and vesicle formation take about 1 min. One single cultured fibloblast cell can produce 2500 coated pits per minute. One example of RME has received a great deal of attention because of its essential role in human health, namely maintaining the proper level of cholesterol in the body. Malfunctions in the RME process for uptake of cholesterol-carrying LDL (see Chapter 14) leads to hypercholesterolemia and cardiovascular disease [11,16]. RME and its role in cholesterol metabolism was discovered by Michael Brown and Joseph Goldstein of The University of Texas Health Science Center in Dallas (now the UT Southwestern Medical Center), who received the 1985 Nobel Prize in Physiology and Medicine for their iconic work.
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4. THE GOLGI Endo-membrane flow, RME, and many other essential cell functions use the Golgi as a central component [17]. The Golgi is often referred to as the “Post Office of the Cell” because its major functions involve packaging and labeling items to be shipped to various destinations around the cell. The Golgi is an intracellular organelle whose large size facilitated its early discovery and morphological description by Italian physiologist Camillo Golgi (Fig. 17.4) in 1897 (see also Chapter 18). After Golgi announced his discovery at a meeting of the Medical Society of Pavia on April 19, 1898, the organelle was named after him. To this day, the Golgi is the only organelle named after a scientist. Discovery of the organelle was facilitated by Golgi’s development of silver as a stain for nerve cells. For his work, Camillo Golgi was awarded the 1906 Nobel Prize in Physiology or Medicine, one of the earliest Nobel Prizes. (In 1901, the first Nobel Prize in Physiology or Medicine went to the German physiologist Emil Adolf van Behring for his work on serum therapy and development of a vaccine against diphtheria.) Golgi’s Italian birth town is now named Corteno Golgi in his honor. The number and size of Golgi vary from cell to cell. Golgi tend to be larger and more numerous in cells that have a large secretion function. Morphologically, the Golgi appears to be composed of membrane-bound stacked structures called cisternae [18]. A “typical” Golgi is depicted in Fig. 17.5. A single mammalian cell usually contains between 40 and 100 Golgi stacks, each of which is composed of 4 to 8 cisternae. Each cisternae is a flat, membrane-enclosed disc that houses the enzymes that conduct the Golgi’s business. The central spaces of the stacked cisternae are contiguous, allowing for processing of cargo as it passes through the Golgi [17]. Each cisternae is composed of four functional regions or networks referred to as the cis-Golgi, medial-Golgi, endo-Golgi, and trans-Golgi. Transport vesicles originating from the ER fuse with the cis-Golgi, releasing their cargo into the lumen of the cisternae. The cargo is modified as it passes through the Golgi, eventually reaching the trans-Golgi, where it is sorted, packaged, and sent to its final destination. Each region of the Golgi contains characteristic enzymes that selectively modify the cargo.
FIGURE 17.4
Camillo Golgi, 1843e1926. http://www.vetmed.vt.edu/education/curriculum/vm8304/lab_companion/ histo-path/vm8054/labs/Lab3/Notes/golginot.htm.
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FIGURE 17.5
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Diagram of a “typical” Golgi. http://www.glogster.com/johnv4297/john-v-s-glog/g-6ltb1bblffg5cerho
4nuna0.
As the cargo proteins pass through the connected Golgi lumen, many modifications are made to ready the proteins for sorting, packaging, and eventual shipment to their appropriate destinations. Most modifications involve glycosylations, primarily to existing sugar chains, although phosphorylations and sulfations are also common. Some of the newly attached sugars may function as a type of signal, directing the protein to the appropriate transport vesicle forming in the trans-Golgi. One modification that has been known for decades involves attaching mannose-6-phosphate, targeting the protein to the lysosome. Sulfation is generally performed by the enzyme sulfotransferase in the trans-Golgi. Added sulfates give the cargo protein a net negative charge that helps in sorting. Cargo proteins destined for secretion or a particular organelle, depart the trans-Golgi in destination-specific transport vesicles and deposit their cargo at the target membrane via fusion.
5. INTRACELLULAR LIPID TRANSPORT While the trafficking of membrane proteins through a cell can usually be followed precisely, the trafficking of membrane lipids is far more troublesome. To begin with for every protein there are 50 or more lipids found in its immediate environment, and these lipids are composed of many different molecular species (see Chapter 10). So, how are membrane lipids transported throughout a cell? While details remain elusive, several overlapping
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mechanisms are possible. Two of these mechanisms, serum albumin and lipoproteins, were discussed in Chapter 14. Other mechanisms include: 1. 2. 3. 4. 5. 6.
Vesicle transport Lipid transfer proteins (LTPs) Lipid lateral diffusion through membranes Free diffusion through the cytosol Membrane to membrane contact Lipid flip-flop
5.1 Lipid Vesicles A living cell is chock-full of lipid vesicles. In fact, there are so many vesicles that for many years cell biologists thought the vesicles were merely artifacts of electron microscopy procedures. A prime example is the secretory vesicles discussed earlier that are involved in membrane trafficking, endo-membrane flow, secretion, and RME. When one of these vesicles fuses with a cell membrane, an enormous number of vesicular membrane lipids are mixed with the cell membrane lipids, increasing the membrane size and altering the membrane lipid composition. This mechanism of lipid transfer likely moves mere lipids than does the other mechanisms. However, vesicle fusion does not have the ability to accurately control the type and amount of lipid transferred.
5.2 Lipid Transfer Proteins LTPs are essential for the movement of lipids both intercellularly and intracellularly and help develop and maintain lipid compositions characteristic of organelles and membrane domains. LTPs were discovered by D.B. Zilversmidt in 1968 as soluble factors that accelerated the transfer of lipids between membranes [19]. We have already encountered an early example of an LTP called a phospholipid exchange protein (PLEP, see Chapter 9). PLEPs have been extensively used to measure transmembrane lipid asymmetry and flip-flop. Since their initial discovery, LTPs have been found in all eukaryotes and bacteria. They come in a wide variety of types, accounting for their ability to transfer many structurally diverse membrane lipids [20,21]. In general, LTPs mediate monomeric lipid exchange in which a single lipid molecule is transported through the aqueous phase sequestered in a hydrophobic pocket of the LTP. Most LTPs are low molecular weight structures that are dominated by b-sheet motifs (Fig. 17.6). A “lid” covers the opening to the hydrophobic pocket and acts as the gate for lipid uptake and release. Thus, LTPs exist in two distinct conformations, a closed conformation that transports the lipid through the aqueous phase and an open conformation where the LTP picks up its lipid cargo at the outer leaflet of a donor membrane and releases it to the outer leaflet of an acceptor membrane. Sterol carrier proteins (also known as nonspecific lipid transfer proteins [NSLTPs]) are a family of LTPs that can transfer a variety of lipids from one intracellular membrane to another [22,23]. They are referred to as “nonspecific” since they have been reported to carry sterols, glycolipids, all common phospholipids, and gangliosides. The sterol carrier proteins
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FIGURE 17.6 Structure of the lipid transfer protein, the sterol carrier protein-2 (SCP-2). http://upload.wikimedia.org/ wikipedia/commons/c/cc/Protein_SCP2_PDB_1c44.png.
are actually two separate proteins: sterol carrier protein X (SCPx, 46 kDa) and the much smaller (13 kDa) sterol carrier protein-2 (SCP-2 is depicted in Fig. 17.6). The SCPs have been suggested to play a role in Zellweger syndrome, a peroxisome disorder. Many other examples of LTPs exist for specific types of lipids: ceramide-transfer protein (CERT), sphingolipid-transfer proteins (CERT and FAPP2), phosphatidylcholine-transfer protein (PCTP), phosphatidylinositol-transfer protein (PITP), retinoid binding proteins (RBPs), and a-tocopherol transfer protein. In mammals, PCTPs, PITPs, and NSLTPs are the three major classes of phospholipid transfer proteins.
5.3 Lipid Lateral Diffusion Through Membranes Although vesicle transfer and LTPs are the most appreciated mechanisms for lipid movement, other significant processes exist. One of the most important of these is lateral diffusion through the membrane plane. Lateral diffusion is responsible for lipid movement through endo-membrane flow. As discussed in Chapter 9, lipids can diffuse rapidly, moving completely around a liver cell in about 1 min. After transport vesicles fuse with a membrane, the newly added lipids rapidly spread out from the site of fusion via lateral diffusion. Lipid lateral diffusion rates are affected by many membrane properties, including lipid bilayer phase, type of lipid micro-domain, lipid heterogeneity, membrane lateral pressure, lipide lipid affinities, lipideprotein affinities, membrane protein crowding, lipid interdigitation, and lipidecytoskeleton interactions (Chapter 10). Lipid vesicle formation is also affected by the membrane lipid content, particularly the acyl chain double bond content, and levels of PS (phosphatidylserine), PA (phosphatidic acid), and DAG (diacylglycerol).
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5.4 Free Diffusion Through the Cytosol Initially, it would seem feasible that membrane lipids could simply be ejected from the bilayer and diffuse across the aqueous compartment to a distant membrane. While theoretically possible, the extremely low solubility of membrane lipids in water would preclude this mechanism from being significant (see Chapter 13, Detergents).
5.5 Membrane-to-Membrane Contact Lipid transfer also occurs at points of contact between distant sites on a membrane.
5.6 Lipid Flip-Flop Finally, lipid transfer is not only associated with diffusion laterally through the membrane plane but also transversely across the membrane in a process known as flip-flop (see Chapter 9). Inherently, lipid flip-flop is a slow event that can be greatly accelerated by proteinelipid interfaces and by enzymes known as flipases, flopases, and scrambleases (see Chapter 9). Many examples of transfer of a specific lipid from one membrane to another for enzymatic modification have been reported [20]. For example, ER PS is transferred to the mitochondria, where it is decarboxylated to PE before being sent back to the ER. Surprisingly, this bidirectional, nonvesicular lipid transfer accounts for most PE synthesis in mammalian cells. Ceramide is also synthesized in the ER and is primarily transported by CERT to the Golgi, where it is converted into sphingomyelin (SM) by SM synthase. It is clear that in cells, membrane lipids are constantly on the move via a variety of mechanisms.
6. SUMMARY A characteristic of all living cells is the continuous movement of material from place to place within the cell. Translocation of membrane-associated constituents is referred to as “membrane trafficking” and forms the heart of the endo-membrane concept. Discussed here are methods commonly used in translocation: secretion (exocytosis), RME, and intracellular carrier proteins. A central component of the endo-membrane flow theory is the Golgi apparatus, a type of molecular processing center that takes in materials from various parts of the cell, modifies and repackages them, and sends them to new cellular locations. Both exocytosis (using SNARE fusion proteins) and endocytosis (through clathrin-coated pits) are largely responsible for the enormous number of intracellular vesicles. Intracellular vesicle fusion to membranes, and a large family of specific and nonspecific LTPs (lipid transfer proteins) are primarily responsible for the rapid movement of polar lipids from one cellular membrane to another. Chapter 18 will discuss involvement of membranes in the essential cell functions of: anesthetic action, G proteinecoupled reactions, membrane attack complex, nerve conduction, and electron transport/oxidative phosphorylation.
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