Chapter 15 • For a cell to operate effectively the different intracellular processes that occur simultaneously must be segregated Membrane enclosed organelles • A prokaryotic cell usually consists of a single compartment, the cytosol enclosed by the plasma membrane • Eukaryotic cells are elaborately subdivided by internal membranes which create a closed compartments in which sets of enzymes can operate without interference from reactions occurring in other compartments • Eukaryotic cells contain a basic set of membrane enclosed organelles  The nucleus is the most prominent organelle, surrounded by a double membrane called the nuclear envelope  The outer nuclear membrane is continuous with the membrane of the ER, which is a major site for synthesis of new membranes in the cells  Small sacs of digestive enzymes called lysosomes degrade worn out organelles and macromolecules (taken in by endocytosis). On the way to lysosomes, endocytosed materials must first pass through a series of compartments called endosomes which sort the ingested molecules and recycle some of them back to the plasma membrane  Many of the membrane enclosed organelles are held in their relative locations in the cell by attachment to the cytoskeleton (microtubules). Cytoskeletal filaments provide tracks for moving the organelles around and for directing the traffic of vesicles between them. These movements are driven by motor proteins that use the energy of ATP hydrolysis to propel the organelles and vesicles along the filaments • Membraneenclosed organelles evolved in different ways  The compartments must have evolved in stages  Membraneenclosed organelles are thought to have arisen in two ways  The nuclear membranes and the membranes of the ER, golgi apparatus, lysosomes and endosomes are believed to have originated by invagination of the plasma membrane.  These membranes and the organelles they enclose are all part of what is collectively called the endomembrane system  The interiors of these organelles communicate with one another and with the outside of the cell by means of small vesicles that bud off from one of these organelles and fuse with another  Mitochondria and chloroplasts are thought to have originated in a different way, they differ from other organelles in that they posses their own small genome and can make some of their own proteins Protein Sorting • Before a eukaryotic cell reproduces by dividing into two, it has to duplicate its membraneenclosed organelles • It requires information and materials contained in the organelle itself so most of the organelles are formed from preexisting organelles that grow and divide • As cells grow, membraneenclosed organelles enlarge by incorporation of new molecules, the organelles then divide and are distributed between daughter cells during cell division. Organelle growth requires a supply of new lipids to make more membrane and a supply of the appropriate proteins • The newly synthesized proteins must be accurately delivered to their appropriate organelle • For some organelles, proteins and lipids are delivered indirectly via the ER, which is itself a major site of lipid and protein synthesis. Proteins enter the ER directly from the cytosol; some are retained there but most are transported by vesicles to the golgi apparatus and then onward to other organelles of plasma membrane • Proteins are imported into organelles by three mechanisms  The fate of any protein molecule synthesized in the cytosol depends on its amino acid sequence, which can contain a sorting signal that directs the protein to the organelle in which it is required  Proteins that lack sorting signals remain as permanent residents in the cytosol  Problem; how can it draw the proteins across membranes that are impermeable to hydrophilic macromolecules  Different ways for different organelles  ONE. Nucleus. Proteins moving from the cytosol to the nucleus move through the nuclear pores that penetrate the inner and outer nuclear membranes. Pores function as selective gates that actively transport specific macromolecules but also allow free diffusion of smaller molecules  TWO. ER, Mitochondria, or Chloroplasts. Proteins are transported across the organelle membrane by protein translocators located in the membrane, the transported protein molecule must usually unfold in order to snake through the membrane  THREE. From the ER onward/ or from one component of the endomembrane system to another. Proteins are transported by a mechanism that is fundamentally different from the other two. The proteins are carried by transport vesicles, which become loaded with a cargo of proteins from the lumen of one compartment as they pinch off from its membrane. The vesicles subsequently discharge their cargo into a second compartment by fusing with its membrane • Signal sequences direct proteins to the correct compartment  The typical sorting signal on proteins is a continuous stretch of amino acid sequence 1560 amino acids long.  The signal sequence is often but not always removed from the finished protein once it has been sorted • Proteins enter the nucleus through nuclear pores  The nuclear envelope forms from two concentric membranes  The inner nuclear membrane contains proteins that act as binding sites for the chromosomes and provide anchorage for the nuclear lamina (a finely woven meshwork of nuclear filaments that lines the inner face of this membrane and provides a structural support for the nuclear envelope  The composition of the outer membrane closely resembles the membrane of the ER, with which it is continuous  The nuclear envelope in all eukaryotic cells is perforated by nuclear pores  Traffic occurs in both directions through the pores; newly made proteins destined for the nucleus enters from the cytosol, RNA molecules synthesized in the nucleus and ribosomal subunits are exported  The nuclear transport serves as a final quality control step in mRNA synthesis and processing  A nuclear pore is a large elaborate structure composed of about 30 different proteins (contain extensive unstructured regions which is thought to form a disordered tangle. The jumbled meshwork fills the center of the channel preventing the passage of large molecules but allowing smaller molecules to pass through). Each pore contains waterfilled passages through which watersoluble molecules can pass freely and non selectively between the cytosol and the nucleus.  Larger molecules must display an appropriate sorting signal to pass through the nuclear pore (nuclear localization signal) that typically consists of one or two short sequences containing several positively charges lysines or arginines  Cyctrolic proteins called nuclear transport receptors bind to the nuclear localization signal on newly synthesized proteins destined for the nucleus and help direct the new protein to a nuclear pore by interacting with the tentaclelike fibrils that extend from the rim of the pore. During transport, the nuclear transport receptors grab onto repeated amino acid sequences within the tangle of nuclear pore proteins pulling themselves from one to the next to carry their cargo protein to the nucleus. once the protein has been delivered the nuclear transport receptor is returned to the cytosol via the nuclear pore for reuse  Importing proteins into the nucleus requires energy (provided by GTP hydrolysis)  Nuclear pore transports proteins in their fully folded conformation and transfer ribosomal components as assembled particles • Proteins unfold to enter mitochondria and chloroplast  Chloroplasts contain a third membrane system, the thylakoid membrane  Some proteins produces in mitochondria and chloroplasts but most protein are encoded by genes in the nucleus, these usually have a signal sequence at their Nterminus that allows them to enter their specific organelles  Proteins are translocated simultaneously across both the inner and outer membranes through specialized sites where the two membranes are in contact with each other.  Each protein is unfolded as it is transported and its signal sequence is removed after translocation is complete  Chaperone proteins inside the organelles help to pull the protein across the two membranes and to refold the protein once it is inside  Subsequent transporting of protein to a particular site inside the organelle requires further sorting signals in the protein, which are exposed after the first signal is removed  Incorporation of new lipids into the membrane is also needed. Most of their membrane phospholipids are thought to be imported from the ER, which is the main site of lipid synthesis in the cell. Phospholipids are transported individually to these organelles by watersoluble lipidcarrying molecules that extract a phospholipids molecule from one membrane and delivers it to another • Proteins enter the endoplasmic reticulum while being synthesized  The ER is the most extensive membrane system in the eukaryotic cell  It serves as an entry point for proteins that are destined for other organelles as well as for the ER itself  Proteins first enter the ER through the cytosol, once inside the ER or embedded in its membrane, individual proteins will now reenter the cytosol through its onward journey they will be carried by transport vesicles from organelle to organelle  Water soluble proteins are completely translocated across the ER membrane and are released into the ER lumen (they are either destined for secretion or for the lumen of an organelle) AND prospective transmembrane proteins are only partly translocated across the ER membrane and are become embedded in it (destined to reside in the ER membrane, the membrane of another organelle, or the plasma membrane)  All these proteins are initially directed to the ER by an ER Signal sequence ( 8 or more hydrophobic amino acids)  Most of the proteins that enter the ER begin to be threaded across the ER before the polypeptide chain has been completely synthesized, this required the ribosome synthesizing the protein to be completely attached to the ER (RER) • Soluble proteins are released into the ER lumen  The ER signal sequence is guided to the ER membrane with the aid of at least two protein components: A signalrecognition particle (SRP) present in the cytosol, which binds to the ER signal when it is exposed on the ribosome AND an SRP receptor , embedded in the membrane of an ER, which recognized the SRP; Binding of an SRP to a signal sequence causes protein synthesis by the ribosome to slow down until the ribosome and its bound SRP locate an SRP receptor in the ER. After binding to the receptor the SRP is released and protein synthesis recommences with the poly peptide now being threaded into the lumen of the ER through a translocation channel in the ER membrane  SRP and SRP receptors function are molecular matchmakers, connecting ribosomes that are synthesizing proteins containing ER signal sequences to available ER translocation channels  The signal sequence is almost always at the Nterminus functions to open the translocation channel • Start and stop signals determine the arrangement of a transmembrane protein in the lipid bilayer  Not all proteins that enter the ER are released to the ER lumen, some remain embedded in the ER membrane as a transmembrane protein  A single membranespanning segment, the Nterminal signal sequence initiates translocation but the transfer process is halted by an additional sequence of hydrophobic amino acids (a stoptransfer sequence) further into the polypeptide chain. This second sequence is released from the translocation channel and drifts into the plane of the lipid bilayer, where it forms and ahelical membranespanning segment that anchors the proteins in the membrane. Simultaneously, the Nterminal signal sequence is also released from the channel into the lipid bilayer and is cleaved off. Transmembrane protein is formed!  In some transmembrane proteins and internal (starttransfer sequence) rather than an Nterminal signal sequence is used to start the protein transfer and is never removed from the polypeptide. This arrangement occurs in some transmembrane proteins in which the polypeptide chain passes back and forth across the lipid bilayer  Hydrophobic signal sequences are thought to work in pairs; an internal starttransfer sequence serves to intiate translocation which continues until a stoptransfer sequence is reached, the two hydrophobic sequences are then released to the bilayer where they remain as membrane spanning alphahelices  In complex multipass proteins, additional pairs of start and stop signal sequences come into play; one sequence reinitiates translocation further down the polypeptide chain while the other stops translocation and causes the polypeptide release and so on for subsequent start and stops Vesicular transport • ER is just the first step • The transport vesicles provide routes of communication between the interior of the cell and its surroundings • They undergo various types of chemical modifications such as the addition of carbohydrate side chains and the formation of disulfide bonds that stabilize protein structure • Transport vesicles carry soluble proteins and Membrane between compartments  Vesicular transport between membraneenclosed compartments of the endomembrane system is highly organized  A major outward secretory pathway starts with the synthesis of proteins on the ER membrane and their entry into the ER and it leads through the golgi apparatus to the cell surface; at the golgi apparatus a side branch leads of through endosomes to lysosomes  A major inward secretory pathway (responsible for ingestion and degradation) moves materials from the plasma membrane, through endosomes to lysosomes  Each transport vesicle that buds off a compartment must take with it only the proteins appropriate to it destination and must fuse only with the appropriate target cell • Vesicle budding is driven by the assembly of a protein coat  Vesicles that bud from membranes usually have a distinctive protein coat on their cytosolic surface and are therefore called coated vesicles  After budding from its parent organelle the vesicle sheds its coat, allowing its membrane to interact directly with the membrane to which it will fuse.  Cells produce several kinds of coated vesicles each with a distinctive protein coat  The coat serves two functions: it shapes the membrane into a bud and it helps to capture molecules for onward transport • Vesicle docking depends on Tethers and SNAREs  After a transport vesicle buds from a membrane, it must find its way to its correct destination to deliver its contents  In most cases, the vesicle is actively transported by motor proteins that move along cytoskeletal fibers  Once a transport vesicle reaches its target, it must recognize and dock with the organelle and then the vesicle membrane can fuse with the target

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