Department of Medical Biochemistry Semmelweis University Subcellular biochemistry February-March 2017
Subcellular biochemistry (biochemical aspects of cell biology) Miklós Csala Semmelweis University Dept. of Medical Chemistry, Molecular Biology and Pathobiochemistry
MOVEMENT AND TRAFFIC OF ORGANELLES Cytoskeleton, microfilaments, microtubuli, actomyosin, vesicular transport
Intracellular traffic diffusion mostly within a compartment protein-mediated transmembrane transport between neighbor compartments channels, carriers, cotransporters, active transporters vesicular transport between detached compartments through the cytosol macromolecules in package pack: microcompartment specificity to contents and target
Vesicular transport endocytosis (PM ) Clathrin-mediated endocytosis (receptor-dependent) Potocytosis (receptor-dependent in caveolae) Macropinocytosis Phagocytosis exocytosis ( PM) constitutive regulated (Ca 2+ triggered) between organelles ER Golgi, Golgi lysosome etc
Vesicular transport Maintained membrane orientation Maintained membrane integrity Luminal remains luminal (or extracellular) Cytosolic remains cytosolic
Formation of the vesicle (budding)
Vesicular transport between cellular organelles
Coat proteins (coatomers) Clathrin COPI COPII endocytosis Golgi plasma membrane (regulated exocytosis) Golgi late endosome Golgi ER (retrograde) Golgi plasma membrane (constitutive exocytosis) ER Golgi (anterograde)
Coat proteins (coatomers) They tend to form polyhedral (e.g. icosahedral, cuboctahedral) lattice.
Triskelion: 3 heavy + 3 light chains Clathrin
Clathrin Adaptins link clathrin to cargo receptors. Polyhedral lattice formation pulls a bud and forms a vesicle. coat disassembly hsc70 (ATPase) fission dynamin (GTPase) Clathrin-coated pit
Fission of clathrin-coated vesicles GTP
COPII-coated vesicles Sar1 (secretion-associated and Ras-related) small GTP binding protein COPI-coated vesicles are assembled similarly by Arf (ADP-ribosylation factor) small GTP binding protein COPII inner coat proteins
COPII-coated vesicles inner coat proteins outer coat proteins Sar1 (or Arf) undergoes autoinactivation by converting GTP to GDP and Pi (GTPase activity). This causes coat depolymerization and yields naked transport vesicles (Sar1-GDP and Arf-GDP are recycled).
Phosphatidylinositol (PI) phosphates as sites for protein attachment PI kinases PI phosphatases
Changing PIP pattern during vesicle transport (not to memorize)
Most steps of vesicle transport are controlled by Rab GTPases Membrane-bound GDI displacement factor GDP dissociation inhibitor Geranylgeranyl group Guanine nucleotide exchange factor Rab cycle GTPase-activating protein SOLUBLE, INACTIVE Rab-GDP MEMBRANE-BOUND, ACTIVE Rab-GTP
Rab effectors and the Rab-controlled steps
Selected Rab GTPases in the vesicle transport pathways of an epithelial cell (not to memorize)
Recognition of the target membrane through SNARE matching NEM: N-Ethylmaleimide NSF: NEM-sensitive factor (an ATPase) SNAP: Soluble NSF attachment protein SNAREs: SNAP receptors
Membrane fusion by SNAREs and SNAPs v-snare SNAP t-snare target membrane NEM: N-Ethylmaleimide NSF: NEM-sensitive factor (ATPase) SNAP: Soluble NSF attachment protein SNAREs: SNAP receptors
Recognition of the target membrane through SNARE matching NEM: N-Ethylmaleimide NSF: NEM-sensitive factor (an ATPase) SNAP: Soluble NSF attachment protein SNAREs: SNAP receptors
Anterograde and retrograde vesicular transport between the ER and Golgi (ERGIC)
Retrieval of ER resident proteins from the Golgi KDEL ER retention signal C-terminal tetrapeptide: lys-asp-glu-leu (KDEL)
Cytoskeleton Cellular protein scaffolding Provides the cell with structure and shape Roles in vesicular transport and cell division Three filaments microfilaments (actin) intermediate filaments microtubules Plasma membrane ER Microtubules Mitochondrion Ribosomes Microfilaments Intermediate filaments
Actin Globular proteins (α,β,γ types) β-actin globular (G) subunit and filamentous (F) polymer polarized polymerization (pointed and barbed ends) function: actomyosin complex (moving organelles, cellular motion cell division), cell adhesion structures, maintenance of cell volume
Dynamic reassembly of actin filaments
Crosslinking / bundling Nucleation / branching
Intermediate filaments No polarity No motor proteins associated High tensile strength Resistant to compression, twisting and bending forces Heterogeneous Types: lamins (nuclear lamina) keratin (epithelial cells, hair, nails) desmin (muscle) vimentin (mesenchymal cells) neurofilaments (neurons) peripherin (some neurons) GFAP (glial fibrillary acidic protein) α-internexin synemin syncoilin nestin phakinin philensin
Microtubules polymers of tubulin subunits alpha and beta-tubulins polarized polymerization (- and + ends) tube formed by 13 polymers (23 nm diameter) negative end at the microtubule organizing center (MTOC): centriole or basal body function: organelle movements, mitotic spindle, cilia and flagella
Microtubules
Microtubules Dynamic instability A: Intermediate concentration of tubulin favors microtubule assembly at the plus end; this end may appear frayed in slow growing filaments. B: At high free GTP-tubulin dimer concentration, hydrolysis is outpaced by rapid assembly at the plus end, thereby forming a rigid GTP-cap. C: Concerted GTP hydrolysis at the plus end weakens the tubulin dimer interaction(s) and the protofilaments rapidly disassemble.
The centrosome
Colchicine "mitotic poison" or spindle poison binds to tubulin and inhibits microtubule polymerization Autumn crocus Colchicum autumnale "meadow saffron"
Motor proteins associated to actin: myosins
Motor proteins associated to microtubules: dyneins and kinesins
Griscelli syndrome disorder characterized by albinism (hypopigmentation) with immunodeficiency GS1: hypopigmentation (blond hair, pale skin, blue sclera), progressive neurological disorder melanosome aggregation in melanocytes myosin Va mutation GS2: all above, plus immunodeficiency hepatosplenomegaly, hypogammaglobulinemia, pancytopenia, recurrent infections, fever Rab27a mutation insufficient exocytosis of cytotoxic granules (lymphocytes, NK cells)