Proteins

Proteins are wedged between the lipids that make up the membrane, and these transmembrane proteins allow molecules that couldn’t enter the cell otherwise to pass through by forming channels, pores or gates. In this way, the cell controls the flow of these molecules as they enter and exit. Proteins in the cell membrane play a role in many other functions, such as cell signaling, cell recognition, and enzyme activity.

In this graphic, the spheres represent the phosphate end, which is polar and water soluble (hydrophilic). The twin extensions represent the fatty acid components which are not water soluble (hydrophobic).

Glycerophospholipids are composed of glycerol, a phosphate group, and two fatty acid chains. 

Glycerol is a three-carbon molecule that functions as the backbone of these membrane lipids.

Within an individual glycerophospholipid, fatty acids are attached to the first and second carbons, and the phosphate group is attached to the third carbon of the glycerol backbone.

Variable head groups are attached to the phosphate. Space-filling models of these molecules reveal their cylindrical shape, a geometry that allows glycerophospholipids to align side-by-side to form broad sheets.

1. Selective permeability. Cell membranes separate the internal and external environments of a cell or organelle, preventing the intrusion of harmful substances, the dispersion of macromolecules, and the dilution of enzymes and substrates. This selective permeability maintains homeostasis. Homeostatic mechanisms attributable to cell membranes maintain optimal intracellular concentrations of ions, water, enzymes, and substrates. Three mechanisms allow selected molecules to cross membranes.

   1. Passive diffusion. Some substances (e.g., water and lipids) can cross the membrane in either direction following a concentration gradient, without the cell expending energy.

   2. Facilitated diffusion. Some molecules (e.g., glucose) are helped across the membrane by a membrane component. This facilitated diffusion is often unidirectional, but it follows a concentration gradient and requires no energy.

   3. Active transport. Some molecules enter or exit a cell against a gradient. This requires energy, usually asadenosine triphosphate (ATP). One active transport mechanism is the sodium pump (Na+/K+-ATPase), which expels sodium ions from a cell even when the sodium concentration is higher outside than inside.

2. Signal transduction. Integral membrane receptor proteins with strong binding affinities for signal molecules (e.g., neurotransmitters, peptide hormones, and growth factors) are found on cell surfaces. The signal molecule to which a receptor specifically binds is its ligand. The receptor transduces the signal across the membrane without the ligand entering the cell. These receptors are critical to intercellular communication (IV.B). Signal transmission depends on the receptor class involved. There are three major receptor classes.

   1. Ligand-gated (e.g., transmitter-gated) ion channelsare long proteins that pass multiple times through the plasma membrane (Fig. 2–2A). The binding of a neurotransmitter (as ligand) to its receptor domain at the cell surface induces a conformational change in the rest of the receptor that opens (or closes) a transmembrane ion channel. Ligand binding and the resulting opening are brief, permitting regulation. A signal of adequate strength (threshold) allows enough ions to cross the membrane to open additional channels (voltage-gated ion channels) in response to the change in potential. Reaching threshold allows a self-perpetuating membrane depolarization to propagate as a wave along the nerve cell surface (9.VII.B.2).

   2. Enzyme-linked receptors (Fig. 2–2B) comprise a heterogeneous group of transmembrane (typically single-pass) proteins associated with an enzyme (typically a protein kinase) or possessing kinase activity of their own (e.g., tyrosine kinase). Protein kinases activate additional proteins by phosphorylating them (III.B.1.b). In this way, a ligand binding to its receptor initiates a cascade of enzyme activations. Receptor tyrosine kinases (e.g., insulinand growth factor receptors) often act by activating an adaptor protein, which subsequently activates the guanosine triphosphate (GTP)-binding protein (G protein) Ras by causing it to exchange the guanosine diphosphate (GDP) it binds in its inactive state for GTP. Activated Ras then activates a cascade of cytoplasmic protein kinases, ending with a gene-regulatory protein that changes the pattern of gene expression. Ras differs from the G proteins described in the next section in that it is monomeric rather than trimeric. It resembles the α subunit in Figure 2–3 and acts without a β or γ subunit.

   3. G protein–coupled receptors (GPCRs) comprise a family of proteins that make seven passes through the membrane (Fig. 2–3). Each receptor binds a different ligand, and the effect of signal binding on the ultimate target protein (either an ion channel [Fig. 2–2A] or anenzyme [Fig. 2–2B]) is indirect and is mediated by a trimeric G protein (Fig. 2–3A). On binding its ligand (Fig. 2–3B), the receptor interacts with and activates the trimeric G protein (Fig. 2–3C). The inactive G protein carries a GDP molecule, which it trades for GTP on activation (Fig. 2–3D and E). The activated G protein complex dissociates from the receptor, allowing the GTP-binding portion to interact with and activate its target (Fig. 2–3F;), thus inactivating the G protein (i.e., GTP → GDP; Fig. 2–3F). Ligand binding to GPCRs results in downstream activation of multiple signaling pathways involving the generation of intracellularsecond messengers. Two key examples with different second messengers are described below.

      1. Cyclic adenosine monophosphate (cAMP).Some adrenalin receptors are key examples of GPCRs that lead to cAMP production. Adrenalin binding activates membrane-bound adenylyl cyclase through a G-protein mediated mechanism, causing the production of cAMPas the second messenger. Among cAMP's primary downstream targets is protein kinase A (PKA), which in turn activates a number of other target molecules including the transcription factors CREB, CREM, and ATF-1.

      2. Diacyl glycerol (DAG) and phosphatidylinositol triphosphate (IP3). Someserotonin, calcitonin, and histamine receptors are GPCRs that lead to DAG and IP3production. Ligand binding activates membrane bound phospholipase C (PLC) through a G-protein mediated mechanism. PLC hydrolyzes the membrane lipid, inositol bisphosphonate (IP2), generating DAG and IP3.

         1. DAG remains in the plasma membrane, facilitating the translocation of protein kinase C (PKC) from the cytosol to the plasma membrane and serving as an activator of that enzyme. PKC then activates other downstream target molecules including MARCK.

         2. IP3 diffuses into the cytosol to bind to IP3-receptors in the smooth endoplasmic reticulum (SER) membranes. IP3-receptors are ligand-gated calcium channels that open upon IP3 binding to allow the release of calcium in the SER into the cytosol. The resulting increase in cytosolic calcium concentration induces the activation of many calcium-dependent processes such as muscle contraction, membrane polarization, and enzyme activity.

   4. Steroid hormone receptor family. Most peptide hormones and growth factors signal the cell interior through cell-surface receptors and associated second messengers. Steroid hormones (e.g., hydrocortisoneand estrogen), retinoids (vitamin A–related compounds), and vitamin D pass more readily through the membrane and bind to receptors in the nucleus. The thyroid hormone receptor also belongs to this group (Because thyroid hormone consists mainly of the aromatic amino, tyrosine, it passes readily through the plasma membrane). This family of nuclear receptors is characterized by a specific ligand-binding site and a DNA-binding site. They typically form dimers and activate or repress gene transcription through their direct association with response elements in the DNA flanking the gene-coding regions

3. Endocytosis. Cells engulf extracellular substances and bring them into the cytoplasm in membrane-limited vesicles by mechanisms described collectively as endocytosis.

   1. In phagocytosis (cell eating), the cell engulfs insoluble substances, such as large macromolecules or entire bacteria. The vesicles formed are termedphagosomes.

   2. In pinocytosis (cell drinking), the cell engulfs small amounts of fluid, which may contain a variety of solutes. Pinocytotic vesicles are smaller than phagosomes.

   3. In receptor-mediated endocytosis (Fig. 2–4), a cell engulfs ligands along with their surface receptors. The binding of ligand to receptor causes the ligand–receptor complexes to collect in a coated pit, a shallow membrane depression whose cytoplasmic surface is covered with the coat protein, clathrin. After further invagination, the protein, dynamin, coils around the neck of the budding vesicle and pinches it off to create a coated vesicle, which carries the ligand–receptor complexes into the cell. The clathrin coat is released from the vesicle, now termed an early endosome, and the ligands dissociate from the receptors. The late endosome, or compartment of uncoupling of receptor and ligand (CURL), becomes more tubular and divides into two portions, segregating the receptors from the ligands. The portion with the receptors pinches off and returns to fuse with the plasma membrane. The portion with the ligands fuses with a lysosome.

4. Exocytosis ejects substances from the cell. Cells use exocytosis both for secretion and for excretion of undigested material. A membrane-limited vesicle or secretory granule fuses with the plasma membrane and releases its contents into the extracellular space, without disrupting the plasma membrane.

5. Compartmentalization. Membranes selectively block the passage of most water-soluble substances. The cytoplasm has many membrane-limited compartments (organelles), each with different internal solute concentrations. This compartmentalization prevents the dilution of substrates, metabolic intermediates and cofactors in multistep biochemical reactions, and protects sensitive reactions from the intrusion of extraneous substances.

6. Spatial–temporal organization of metabolic processes. Some cell membranes (e.g., inner mitochondrial membrane and Golgi complex) contain enzymes arranged in series so that intermediates in multistep metabolic processes are passed fromenzyme to enzyme. This arrangement maintains the chronologic order of such processes and sets rate limits by maintaining local concentrations of intermediates.

7. Storage, transport, and secretion. Substances in vesicles may be kept for later use (storage), shuttled from one compartment to another for further processing (transport, II.D), or expelled from the cell (secretion, II.C.4).

D. Membrane Flow (Traffic)

Membrane moves from one organelle to another. This general feature of organelle function allows homeostatic regulation of membrane area. Membranes bud as vesicles from one organelle and fuse with another, allowing the amount of membrane in a particular organelle to change without membrane synthesis or breakdown. Vesicle budding is energetically facilitated by coat proteins (e.g., β-COP, clathrin) that vesicles acquire on their cytoplasmic surfaces during budding. These coats help shape the bud and ensure its inclusion of molecules that direct vesicle transport toward the proper destination. After a coated vesicle is formed, the coat is released. The resulting transfer vesiclemay diffuse to nearby targets (e.g., ER to Golgi) or attach to microtubules for transport over longer distances (e.g., neuron cell body to axon terminal). Transmembrane proteins in budding vesicle membranes, called v-SNAREs, remain after the coat is shed. They are critical in allowing transfer vesicles to recognize, bind, fuse with, and unload their contents, in a GTP-dependent process, at the correct target organelle. Receptors in the target organelle membranes, called t-SNAREs,selectively bind the v-SNAREs and recruit other cytoplasmic proteins required for fusion with the target membrane.

(a) Membranes of animal cells have as their major lipid components phospholipids and cholesterol. A phospholipid is amphipathic, with a phosphate group charge on the polar head and two long, nonpolar fatty acid chains, which can be straight (saturated) or kinked (at an unsaturated bond). Membrane cholesterol is present in about the same amount as phospholipid.

(b) The amphipathic nature of phospholipids produces the bilayer structure of membranes as the charged (hydrophilic) polar heads spontaneously form each membrane surface, in direct contact with water, and the hydrophobic nonpolar fatty acid chains are buried in the membrane’s middle, away from water. Cholesterol molecules are also amphipathic and are interspersed less evenly throughout the lipid bilayer; cholesterol affects the packing of the fatty acid chains, with a major effect on membrane fluidity. The outer layer of the cell membrane also contains glycolipids with extended carbohydrate chains.

Sectioned, osmium-fixed cell membrane may have a faint trilaminar appearance with the transmission electron microscope (TEM), showing two dark (electron-dense) lines enclosing a clear (electron-lucent) band. Reduced osmium is deposited on the hydrophilic phosphate groups present on each side of the internal region of fatty acid chains where osmium is not deposited. The “fuzzy” material on the outer surface of the membrane represents the glycocalyx of oligosaccharides of glycolipids and glycoproteins. (X100,000)

(a) The fluid mosaic model of membrane structure emphasizes that the phospholipid bilayer of a membrane also contains proteins inserted in it or associated with its surface (peripheral proteins) and that many of these proteins move within the fluid lipid phase. Integral proteins are firmly embedded in the lipid layers; those that completely span the bilayer are called transmembrane proteins. Hydrophobic amino acids of these proteins interact with the hydrophobic fatty acid portions of the membrane lipids. Both the proteins and lipids may have externally exposed oligosaccharide chains.

(b) When cells are frozen and fractured (cryofracture), the lipid bilayer of membranes is often cleaved along the hydrophobic center. Splitting occurs along the line of weakness formed by the fatty acid tails of phospholipids. Electron microscopy of such cryofracture preparation replicas provides a useful method for studying membrane structures. Most of the protruding membrane particles seen (1) are proteins or aggregates of proteins that remain attached to the half of the membrane adjacent to the cytoplasm (P or protoplasmic face). Fewer particles are found attached to the outer half of the membrane (E or extracellular face). Each protein bulging on one surface has a corresponding depression (2) on the opposite surface.

Both protein and lipid components often have covalently attached oligosaccharide chains exposed at the external membrane surface. These contribute to the cell’s glycocalyx, which provides important antigenic and functional properties to the cell surface. Membrane proteins serve as receptors for various signals coming from outside cells, as parts of intercellular connections, and as selective gateways for molecules entering the cell.

Transmembrane proteins often have multiple hydrophobic regions buried within the lipid bilayer to produce a channel or other active site for specific transfer of substances through the membrane.

Studies with labeled membrane proteins of cultured cells reveal that many such proteins are not bound rigidly in place and are able to move laterally (Figure 2–4). Such observations as well as data from biochemical, electron microscopic, and other studies showed that membrane proteins comprise a moveable mosaic within the fluid lipid bilayer, the well-established fluid mosaic model for membrane structure (Figure 2–2a). Unlike the lipids, however, lateral diffusion of many membrane proteins is often restricted by their cytoskeletal attachments. Moreover, in most epithelial cells tight junctions between the cells (see Chapter 4) also restrict lateral diffusion of unattached transmembrane proteins and outer layer lipids, producing different domains within the cell membranes.

+

Download Slide (.ppt) | Print

(a) Two types of cells were grown in tissue cultures, one with fluorescently labeled transmembrane proteins in the plasmalemma (right) and one without.

(b) Cells of each type were fused together experimentally into hybrid cells.

(c) Minutes after the fusion of the cell membranes, the fluorescent proteins of the labeled cell spread to the entire surface of the hybrid cells. Such experiments provide important data supporting the fluid mosaic model. However, many membrane proteins show more restricted lateral movements, being anchored in place by links to the cytoskeleton.

+

• Diffusion transports small, nonpolar molecules directly through the lipid bilayer. Lipophilic (fat-soluble) molecules diffuse through membranes readily, water very slowly.

• Channels are multipass proteins forming transmembrane pores through which ions or small molecules pass selectively. Cells open and close specific channels for Na+, K+, Ca2+ and other ions in response to various physiological stimuli. Water molecules usually cross the plasma membrane through channel proteins called aquaporins.

• Carriers are transmembrane proteins that bind small molecules and translocate them across the membrane via conformational changes.

Major mechanisms by which molecules cross membranes.

Graphic Jump Location

+

Download Slide (.ppt) | Print

Lipophilic and some small, uncharged molecules can cross membranes by simple diffusion (a).

Most ions cross membranes in multipass proteins called channels (b) whose structures include transmembrane ion-specific pores.

Many other larger, water-soluble molecules require binding to sites on selective carrier proteins (c), which then change their conformations and release the molecule to the other side of the membrane.

Diffusion, channels and most carrier proteins translocate substances across membranes using only kinetic energy. In contrast, pumps are carrier proteins for active transport of ions or other solutes and require energy derived from ATP.

Mechanisms of transport across the plasma membrane.

The cell membrane separates the interior of cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells.

It consists of the phospholipid bilayer with embedded proteins.

Cell membranes are involved in a variety of cellular processes such as cell adhesion, ion conductivity and cell signalling and serve as the attachment surface for several extracellular structures, including the cell wall, glycocalyx, and intracellular cytoskeleton. Cell membranes can be artificially reassembled.

The limiting membranes that envelop all eukaryotic cells are made of phospholipids, cholesterol, proteins, and oligosaccharide chains covalently linked to phospholipid and protein molecules.

The cell membrane functions as a selective barrier regulating the passage of materials into and out of the cell and facilitating the transport of specific molecules. One important role of the cell membrane is to keep constant the ion content of cytoplasm, which differs from that of the extracellular fluid. Membranes also carry out a number of specific recognition and signaling functions, playing a key role in the interactions of the cell with its environment.The plasma membrane defines the outer limit of the cell. However a continuum exists between the interior of the cell and extracellular macromolecules. The plasma membrane contains proteins called integrins linked to both cytoplasmic protein filaments and ECM components. These linkages produce a continuous exchange of influences, in both directions, between the ECM and the cytoplasm.¹

Membranes range from 7.5 to 10 nm in thickness and consequently are visible only in the electron microscope.

Membrane phospholipids are amphipathic, consisting of two nonpolar (hydrophobic or water-repelling) long-chain fatty acids linked to a charged polar (hydrophilic or water-attracting) head that bears a phosphate group.

Figure: Lipids in membrane structure.

Phospholipids are most stable when organized into a double layer (bilayer) with the hydrophobic fatty acid chains directed toward the middle away from water and the hydrophilic polar head groups facing the water. Molecules of cholesterol, a sterol lipid, insert at varying densities among the closely-packed phospholipid fatty acids, restricting their movement, and modulating the fluidity and movement of all membrane components. The phospholipids in each half of the bilayer are different. For example, in the well-studied membranes of red blood cells phosphatidylcholine and sphingomyelin are more abundant in the outer half, while phosphatidylserine and phosphatidylethanolamine are more concentrated in the inner layer. Some of the outer lipids, known as glycolipids, include oligosaccharide chains that extend outward from the cell surface and contribute to a delicate cell surface coating called the glycocalyx. With the transmission electron microscope (TEM) the cell membrane—and all other organellar membranes—may exhibit a trilaminar appearance after fixation in osmium tetroxide; osmium binding the polar heads of the phospholipids, the outer sugar chains, and associated membrane proteins produces the two dark outer lines enclosing the light band of osmium-free fatty acids.

Figure 2–2 Proteins associated with the membrane lipid bilayer.

(a) The fluid mosaic model of membrane structure emphasizes that the phospholipid bilayer of a membrane also contains proteins inserted in it or associated with its surface (peripheral proteins) and that many of these proteins move within the fluid lipid phase. Integral proteins are firmly embedded in the lipid layers; those that completely span the bilayer are called transmembrane proteins. Hydrophobic amino acids of these proteins interact with the hydrophobic fatty acid portions of the membrane lipids. Both the proteins and lipids may have externally exposed oligosaccharide chains.

(b) When cells are frozen and fractured (cryofracture), the lipid bilayer of membranes is often cleaved along the hydrophobic center. Splitting occurs along the line of weakness formed by the fatty acid tails of phospholipids. Electron microscopy of such cryofracture preparation replicas provides a useful method for studying membrane structures. Most of the protruding membrane particles seen (1) are proteins or aggregates of proteins that remain attached to the half of the membrane adjacent to the cytoplasm (P or protoplasmic face). Fewer particles are found attached to the outer half of the membrane (E or extracellular face). Each protein bulging on one surface has a corresponding depression (2) on the opposite surface.

• Structure
• Function