Intracellular Compartments and Protein Transport PDF

CHAPTER 15 Intracellular Compartments and Protein Transport After the completion of this content, students will be able to: 1) Describe the structure and function of intracellular compartments. 2) Explain the mechanisms of protein transport between intracellular compartments. 3) Describe the...

CHAPTER 15 Intracellular Compartments and Protein Transport After the completion of this content, students will be able to: 1) Describe the structure and function of intracellular compartments. 2) Explain the mechanisms of protein transport between intracellular compartments. 3) Describe the mechanisms of protein trafficking and secretion. Copyright © 2023 by W. W. Norton & Company, Inc.  Intracellular Compartments and Transport At any one time, a typical eukaryotic cell carries out thousands of different chemical reactions, many of which are mutually incompatible. For example, if the cells of an organ, such as the liver, are broken apart and their contents mixed together in a test tube, chemical chaos results, and the cells’ enzymes and other proteins are quickly degraded by their own proteolytic enzymes. For a cell to operate effectively, the different intracellular processes that occur simultaneously must somehow be segregated. Two strategies for isolating and organizing different chemical reactions: 1: Both prokaryotic and eukaryotic cells aggregate different enzymes required to catalyze a particular sequence of reactions into large, multicomponent complexes, such as synthesis of DNA, RNA, and proteins. 2: Most highly developed in eukaryotic cells, it is to confine different metabolic processes and the proteins required to perform them, within different membrane-enclosed compartments. In eukaryotic cells: membrane-enclosed compartments are called membrane- enclosed organelles.  (I) Membrane-enclosed organelles  Intracellular Compartments / Organelles In eukaryotic cells, internal membranes create enclosed compartments and organelles in which different metabolic processes are segregated. An intestinal cell contains the basic set of organelles  Main functions of membrane-enclosed organelles Nucleus is generally the most prominent organelle, which is surrounded by a double membrane known as nuclear envelope and communicates with the cytosol via nuclear pores that perforate the envelope. Endoplasmic reticulum (ER) is the major site for synthesis of new membranes. Rough ER: Having ribosomes attached to its cytosolic surface Smooth ER: Lacking ribosomes ER performs particular functions: Adrenal gland cells: steroid hormone synthesis liver cells: detoxify alcohol smooth ER: sequester Ca2+ from the cytosol and the release of Ca2+ to cytosol triggers secretion of signal molecules and the contraction of muscle cells Golgi apparatus: receives proteins and lipids from the ER, modifies them, and then dispatches them to other destinations in the cells. Lysosomes: small sacs of digestive enzymes degrade worn-out organelles, as well as macromolecules and particles taken into the cell by endocytosis. Endosomes: compartments containing endocytosed materials. Peroxisomes: small organelles with a single membrane contain enzymes used in a variety of oxidative reactions that break down lipids and destroy toxic molecules. Mitochondria: pyruvate oxidation, citric acid cycle, and oxidative phosphorylation Chloroplasts: photosynthesis Relative volumes of membrane-enclosed organelles  The evolution of membrane-enclosed organelles likely began with an expansion of the plasma membrane An ancient archaeon cell possibly enlarged its plasma membrane, forming protrusions allowing it to interact with aerobic bacteria Protrusions invaginated giving rise to various membrane-enclosed organelles by enclosing the archaeon’s genetic material, engulfing aerobic bacteria, and fusing to form the components of the endomembrane system (ER and Golgi apparatus) This can explain why the nucleus in present-day eukaryotes is surrounded by a double-layered membrane, and why mitochondria have two membranes, their own genomes, and do not participate in vesicular transport of the endomembrane system  (II) Protein sorting Membrane-enclosed organelles import proteins by one of three mechanisms The synthesis of virtually all proteins in the cell begins on ribosomes in the cytosol. The exceptions are the few mitochondrial and chloroplast proteins that are synthesized on ribosomes inside these organelles; most mitochondrial and chloroplast proteins are made in the cytosol and subsequently imported. Sorting signal directs the protein to the organelle in which it is required. (Proteins that lack such signals remain as permanent residents in the cytosol ) 1: Transport through nuclear pores: proteins moving from the cytosol into the nucleus are transported through the nuclear pores that penetrate the inner and outer nuclear membranes. 2: Transport across membranes: proteins moving from the cytosol into the ER (resident vs non-resident proteins), mitochondria, or chloroplast are transported across the organelle membrane by protein translocators located in the membrane. (Unfolding is required for protein transport across the membranes) 3: Transport by vesicles: transport vesicles are loaded with a cargo of proteins from the lumen of one compartment of the endomembrane system (ER, Golgi apparatus, endosomes and lysosomes), and discharge their cargo into a second compartment by membrane fusion. Signal sequences that direct proteins to the correct compartment  Signal sequences direct proteins to the correct organelle (A) Proteins destined for the ER posses an N-terminal signal sequence that directs them to that organelle, whereas those destined to remain in the cytosol lack this sequence. (B) If the signal sequence is removed from the ER protein and attached to the cytosolic protein, the proteins end up in an abnormal location in the cell, indicating the ER signal sequence is both necessary and sufficient to direct a protein to the ER. The outer nuclear membrane is continuous with the ER The double membrane of the nuclear envelope is penetrated by nuclear pores. The nuclear envelope encloses the nuclear DNA and defines the nuclear compartment. Traffic occurs in both directions though the pores: Newly made proteins destined for the nucleus enter from the cytosol RNA molecules, which are synthesized in the nucleus, and ribosomal subunits, which are assembled in the nucleus, are exported. The nuclear pore complex forms a gate through which molecules enter or exit from the nucleus The inner nuclear membrane contains proteins that act as binding sites for the chromosomes and provide anchorage for the nuclear lamina. A nuclear pore is composed of about 30 different proteins. Each pore contains water- filled passages through which small water-soluble molecule can pass freely. Many proteins that line the nuclear pore contain extensive and unstructured regions, preventing the passage of large molecules. Proteins bound for the nucleus are actively transported through nuclear pores Nuclear localization signal: the signal sequence that directs a protein from the cytosol into the nucleus typically consists of one or two short sequences containing several positively charged lysines or arginines. Nuclear transport receptors bind to the nuclear localization signal on newly synthesized proteins destined for the nucleus. These receptors help direct the new protein through the nuclear pore into the nucleus. Once the protein has been delivered, the nuclear transport receptor is returned to the cytosol via the nuclear pore for reuse. The energy supplied by GTP hydrolysis drives nuclear transport 1. A nuclear transport receptor picks up its cargo protein in the cytosol and enters the nucleus. 2. In the nucleus, Ran-GTP binds to the nuclear transport receptor, causing it to release its cargo. 3. The nuclear transport receptor - still carrying the Ran-GTP- is transported back through the pore to the cytosol. 4. In the cytosol, an accessory protein triggers Ran to hydrolyze its bound GTP to GDP. Ran- GDP falls off the nuclear transport receptor, which is then free to bind another cargo protein destined for the nucleus. A similar cycle operates to export mRNAs and other large molecules from the nucleus into the cytosol. Movie: Nuclear Import and Export  Proteins are imported into mitochondrial in an unfolded form The mitochondrial signal sequence of a precursor protein is recognized by a receptor in the outer mitochondrial membrane. The complex of receptor and attached protein diffuses laterally in the membrane to a contact site, where the protein is translocated across both the outer and inner membranes by a protein translocator. The signal sequence is cleaved off by a signal peptidase inside the mitochondrion. The chaperone proteins that help to pull the protein across the membranes and help it to refold are not shown. Movie: Mitochondrial protein import  The endoplasmic reticulum is the most extensive membrane network in eukaryotic cells (A) Fluorescence micrograph of a living plant cell showing the ER as complex network of tubes. The cell contains a fluorescent protein in its ER. (B) An electron micrograph showing the rough ER in a cell from a dog’s pancreas, which makes and secretes large amounts of digestive enzymes. The cytosol is filled with closely packed rough ER. A common pool of ribosomes is used to synthesize both the proteins that stay in the cytosol and the ER Two separate populations of ribosomes in the cytosol Membrane-bound ribosomes: attached to the cytosolic side of the ER membrane and are making proteins that are being translocated into ER. Free ribosomes: unattached to any membrane and are making all of the other proteins encoded by the nuclear DNA. Many ribosomes bind to each mRNA molecule, forming a polyribosome. At the end of each round of protein synthesis, the ribosomal subunits are released and rejoin the common pool in the cytosol.  An ER signal sequence and a SRP direct a ribosome to the ER membrane The Signal-Recognition Particle (SRP) binds to the exposed ER signal sequence and to the ribosome, thereby slowing protein synthesis by the ribosome. The SRP-ribosome complex then binds to an SRP receptor in the ER membrane. The SRP is released, passing the ribosome to a translocation channel in the ER membrane. Finally, the translocation channel inserts the polypeptide chain into the membrane and starts to transfer it across the lipid bilayer.  A soluble protein crosses the ER membrane and enters the lumen A translocation channel binds the signal sequence and actively transfers the rest of the polypeptide across the lipid bilayer as a loop. During the translocation process, the signal peptide is cleaved from the growing protein by a signal peptidase. The cleaved signal sequence is ejected into the bilayer, where it is degraded, and the translocated polypeptide is released as soluble protein into the ER lumen. The channel closes once the protein has been released.  A single-pass transmembrane protein is integrated into the ER membrane An N-terminal ER signal sequence (hydrophobic start-transfer sequence) initiates transfer of the protein (also contains a second hydrophobic sequence, a stop-transfer sequence) When this sequence enters the translocation channel, the channel discharges the protein sideways into the lipid bilayer. The N-terminal signal sequence is cleaved off, leaving the transmembrane protein anchored in the membrane. Protein synthesis on the cytosolic side continues to completion. *Membrane-bound portion of protein is usually an α-helix*  A double-pass transmembrane protein uses an internal start-transfer sequence to integrate into the ER membrane An internal ER signal sequence acts as a start-transfer signal and initiates the transfer of the polypeptide chain. Like the N-terminal ER signal sequence, the internal start-transfer signal is recognized by an SRP that brings the ribosome to the ER membrane. When a stop-transfer sequence enters the translocation channel, the channel discharges both sequences into the membrane. Neither the start- transfer nor the stop- transfer sequence is cleaved off and the entire polypeptide chain remains anchored in the membrane as a double-pass transmembrane protein. Proteins that span the membrane more times contain further pairs of stop and start sequences, and the same process is repeated for each pair. Movie: Protein Translocation into the ER Re-cap: Intracellular Compartments and Transport Eukaryotic membrane-enclosed organelles: nucleus, ER, Golgi apparatus, lysosomes, endosomes, mitochondria, and peroxisomes. Most organelle proteins are made in the cytosol and transported into the organelle. Sorting signals guide the proteins to the correct organelle; proteins that function in the cytosol have no such signals and remain in the cytosol. Nuclear proteins contain nuclear localization signals that direct their nuclear import through nuclear pore complexes without being unfolded. Most mitochondrial proteins are made in the cytosol and must be unfolded to allow them to pass through the protein translocators on the membrane. The ER is the membrane factory of cell; it makes most of the cell’s lipids and many of its proteins. Ribosomes in the cytosol are directed to the ER if the protein that they are making has an ER signal sequence, which is recognized by a signal-recognition particle (SRP) in the cytosol; the binding of ribosome-SRP complex to a SRP receptor on the ER membrane initiates the translocation process through the ER membrane. Soluble proteins destined for secretion or for the lumen of an organelle pass completely into the ER lumen, while transmembrane proteins destined for the ER membrane or for other cell membranes remain anchored in the lipid bilayer by one or more membrane-spanning α helices.  (III) Vesicular Transport  Transport vesicles carry soluble protein and membrane between cell compartments Vesicular transport between membrane-enclosed compartments of endomembrane system (ER, Golgi apparatus, endosomes and lysosomes) is highly organized. In the outward Secretory Pathway, the synthesized proteins are transported from ER, through Golgi apparatus, to plasma membrane, or via early and late endosomes to lysosomes. In the inward endocytic pathway, extracellular molecules are ingested in vesicles derived from the plasma membrane and are delivered to early endosomes and then via late endosomes to lysosomes. Each compartment encloses a space, or lumen, that is topologically equivalent to the outside of the cell. Clathrin forms basketlike cage that help shape membranes into vesicles  Electron micrographs showing numerous clathrin- coated pits and vesicles budding from the inner surface of the plasma membrane of cultured skin cells.  At the plasma membrane, each vesicle starts off as a clathrin-coated pit.  These clathrin-coated vesicles bud from the plasma membrane on the inward endocytic pathway and from the Golgi apparatus on the outward secretory pathway. Clathrin-coated vesicles transport selected cargo molecule Cargo receptors, with their bound cargo molecules, are captured by adaptins, which also bind clathrin molecules to the cytosolic surface of the budding vesicle. Dynamin proteins assemble around the neck of budding vesicles, hydrolyze their bound GTP, and pinch off the vesicle. After budding is complete, the coat proteins are removed, and the naked vesicle can fuse with its target membrane. Rab proteins and SNAREs direct transport vesicles to target membrane A filamentous tethering protein on a membrane binds to a Rab protein on the surface of a vesicle. This interaction allows the vesicle to dock on its target membrane. A v-SNARE on the vesicle then binds to a complementary t-SNARE on the target membrane. Whereas Rab and tethering proteins provide the initial recognition between a vesicle and its target membrane, the pairing of complementary SNAREs also helps ensure that transport vesicles reach their appropriate target membrane.  SNARE proteins play a central role in membrane fusion Pairing of v-SNAREs and t-SNAREs draw the two lipid bilayers into close proximity The force of the SNAREs winding together squeezes out any water molecules between the two membranes, allowing their lipids to flow together to form a continuous bilayer. Additional proteins help to pry the SNAREs apart so they can be used again. Movie: Clathrin-coated vesicles  (IV) Secretory Pathways  Many proteins are glycosylated in the ER Exocytosis: newly made proteins, lipids, and carbohydrates are delivered from the ER, via the Golgi apparatus, to the cell surface by transport vesicles that fuse with plasma membrane. As soon as the polypeptide chain enters the ER lumen, its is glycosylated by the addition of oligosaccharide side chains to the NH2 region of an asparagine in the polypeptide (called N- linked oligosaccharide side chains, the most common type of linkage found on glycoprotein). Each oligosaccharide chain is transferred as an intact unit to the asparagine from a lipid, catalyzed by a membrane-bound oligosaccharide protein transferase with its active site exposed on the lumenal side of the ER membrane. This explains why cytosolic proteins are not glycosylated in this way.  Chaperones prevent misfolded or partially assembled proteins from leaving the ER Misfolded proteins bind to chaperone proteins in the ER lumen and are retained, whereas normally folded proteins are transported in transport vesicles to the Golgi apparatus. If the misfolded proteins fail to refold normally, they are transported back to the cytosol, where they are degraded.  Misfolded proteins in the ER lumen trigger the production of chaperones and the expansion of the ER Misfolded proteins bind to receptors that stimulate the production of a transcriptional regulator. This protein translocates to the nucleus where it activates genes that encode chaperones and other ER components, thus promoting the proper folding and processing of proteins. The system is known as the unfolded protein response (UPR). The Golgi apparatus is made of a stack of membrane-enclosed sacs Each Golgi stack has two distinct faces: an entry, or cis, face and an exit, or trans, face. (The cis face is adjacent to the ER, while the trans face points toward the plasma membrane) Soluble proteins and membrane enter the cis Golgi network via transport vesicles derived from the ER. The proteins travel through the cisterna in sequence by means of transport vesicles that bud from one cisterna and fuse with the next. Proteins exit from the trans Golgi network via transport vesicles destined for either the cell surface or another compartment. In secretory cells, the regulated and constitutive pathways of exocytosis diverge in the trans Golgi network Many soluble proteins are continually secreted from the cell by the constitutive secretory pathway, which operates in all cells. This pathway also continually supplies the plasma membrane with newly synthesized lipids and proteins. The regulated exocytosis pathway operates only in cells that are specialized for secretion. Specialized secretory cells produce large quantities of particular products, such as hormones, mucus, or digestive enzymes, which are stored in secretory vesicle, where the proteins are concentrated and stored until an extracellular signal stimulates their secretion.  (V) Endocytic Pathways  Phagocytic cell ingest other cell Two main types of endocytosis are distinguished on the basis of the size of the endocytic vesicles formed: Pinocytosis (cellular drinking) involves the ingestion of fluid and molecules via small vesicles (250 nm in diameter) (A) Electron micrograph of a phagocytic white blood cell (a neutrophil) ingesting a bacterium, which is in the process of dividing. (B) Scanning electron micrograph showing a macrophage engulfing a pair of red blood cells.  LDL enters cells via receptor-mediated endocytosis Cholesterol is extremely insoluble and is transported in the bloodstream bound to protein in the form of particles call low-density lipoproteins, or LDL. The LDL binds to receptors on the cell surface and internalized in clathrin-coated vesicle. The vesicles lose their coat and then fuse with endosomes. In the acidic environment of the endosome, LDL dissociates from its receptors. Whereas the LDL ends up in lysosomes, where it is degraded to release free cholesterol, the LDL receptors are returned to the plasma membrane via transport vesicles to used again. Whether it is occupied or not, an LDL receptor typically makes one round trip into the cell and back every 10 minutes, making a total of several hundred trips in its 20-hour life span. Movie: receptor-mediated endocytosis The fate of the receptor proteins involved in endocytosis depends on the type of receptor Three pathways: recycling: to the same plasma membrane degradation: go to lysosome transcytosis: different domain of plasma membrane A lysosome contains hydrolytic enzymes and an H+ pump Materials destined for degradation follow different pathways to the lysosome Autophagy: degrading obsolete parts of the cell itself. The process begins with enclosure of the organelle by a double membrane, creating an autophagosome, which is then fuses with lysosome.