5 OverviewBriefly describe membrane-enclosed organelles of eukaryotic cells and their functionsDiscuss how the protein composition of each organelle/compartment is formed and maintainedDiscuss how organelles communicate with each otherVesicles
11 Protein SortingHaving briefly reviewed the main membrane-enclosed organelles of the eukaryotic cell, we turn now to the question of how each organelle acquires its unique set of proteins.
12 How are proteins sorted into discrete locations? Cell must duplicate its membrane-enclosed organelles before dividingMost organelles formed from preexisting organelles then divide and are distributed between daughter cellsNon-dividing cells continuously generate proteins and replace proteins that have been degradedProteins need to be sorted correctlyorganelle membrane proteins, organelle lumen proteins, secreted proteinsHOW? 3 Mechanisms
28 2 kinds of proteins transferred from the cytosol to the ER Water-soluble proteins - translocated across ER membrane into the ER lumenDestined for secretion or for the lumen of an organelleProspective transmembrane proteins - partially translocated across ER membrane and become embedded in itDestined to stay in ER membrane, membrane of another organelle, or plasma membraneDirected to ER by ER signal sequence - 8 or more hydrophobic amino acids
35 Multi-pass Transmembrane Proteins Need additional pairs of stop and start sequencesOne sequence reinitiates translocation further down peptide chainThe other stops translocation and causes polypeptide release and so onStitched into membrane as they are synthesized
37 Two types – secretory pathway and endocytic pathway Secretory pathway Vesicular TransportTwo types – secretory pathway and endocytic pathwaySecretory pathwayEntry into the ER is the first step - pathway to another destinationInitial destination is the Golgi apparatusFrom Golgi to other compartments - carried out by budding and fusion of transport vesiclesExtend outward: ER plasma membraneEndocytic pathwayExtend inward: plasma membrane lysosomesCOMMUNICATION between cells!
39 Vesicle Budding – Assembly of a Protein Coat Vesicles that bud from membranes have a distinctive protein coat on cytosolic surface - coated vesiclesAfter budding from parent organelle - sheds the coat allowing the vesicle to interact directly with the membrane it will fuse toCells produce different types of coatedvesicles – focus on ClathrinTwo functions of the coat:Shapes the membrane into a budHelps capture molecules for onward transport
47 Intracellular bacteria Chlamydia spp.MycobacteriumSalmonellaWHY? Reduced genome sizes compared to extracellular bacteriaE. coli = 4.6 megabasesChlamydia = 1.3 megabasesDo not have the genes to synthesize many essential nutrients – e.g. Amino acidsMust parasitize these from their host!Take advantage of vesicular trafficking and hijack nutrient-rich vesicles!
48 Many intracellular bacteria target Rab Proteins! Mycobacterium tuberculosis acquires iron and other nutrients by targeting recycling endosome and trans-Golgi Rab ProteinsChlamydia spp. acquire sphingolipids and amino acids by targeting trans-Golgi Rab ProteinsUninfected human epithelium cells (left) with compact Golgi band close to the cell nucleus (blue) and cells infected with Chlamydiae trachomatis with Golgi fragments (red) which accumulate around the bacterial inclusion (green).
49 How do proteins cross the plasma membrane? Secretory Pathways
50 ExocytosisExocytosis - 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 the plasma membraneFIXED sequence of membrane-enclosed compartments - often chemically modified en route
51 Chemical modifications which occur in the ER Disulfide bonds are formed between cysteine side chainsRemember? Stabilize protein structureGlycosylation - covalent attachment of short oligosaccharide side chainsRemember? Protect extracellular proteins, serve as transport signal form carbohydrate layer, help with cell to cell recognitionCarried out by glycosylating enzymes in ER but not in the cytosol
55 Proteins further modified and sorted in the Golgi apparatus Oligosaccharides added in ER are further modifiedremoving or adding sugarsGolgi apparatusUsually located near cell nucleusCollection of flattened membrane-enclosed sacs called cisternae with two distinct faces:Cis face is adjacent to the ERTrans face points toward the plasma membraneOutermost cisterna on each face is connected to a network of tubules and transport vesicles
60 How do proteins enter the cell? Endocytic Pathways Review video:
61 EndocytosisEndocytosis - eukaryotic cells continuously take up fluid, as well as large and small moleculesMaterial to be ingested enclosed by a small portion of plasma membrane - buds inward and then pinches off to form an intracellular endocytic vesicle
62 2 main types of Endocytosis Pinocytosis (“cellular drinking”) - ingestion of fluid and molecules via small vesicles (< 150nm in diameter)Carried out predominantly by clathrin-coated vesiclesPhagocytosis (“cellular eating”) - ingestion of large particles via large vesicles called phagosomes (generally > 250nm in diameter)Only “phagocytic” cells do this
64 Receptor-mediated Endocytosis Pinocytosis is indiscriminateReceptor-mediated Endocytosis = controlled pinocytosisMore efficient - macromolecules bind to complementary receptors on the cell surface and enter the cell as receptor-macromolecule complexes in clathrin-coated vesiclesSelective concentrating mechanismIncreases efficiency of internalization of particular macromolecules more than 1000-fold compared to pinocytosisExample: Cholesterol
66 Endosomes – site for sorting of imported macromolecules Two sets of endosomesEarly endosomes: Just beneath plasma membrane - mature into late endosomes as they fuse with each otherLate endosomes: Closer to the nucleusInterior of endosome kept acidic by proton pump in the endosomal membranepH 5-6Low pH causes receptors to release their cargoMain sorting station in the inward endocytic pathwaytrans-Golgi is the sorting station for exocytic or secretory pathway!