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Traffic to and Function of Organelles

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Traffic to and Function of Organelles. A. Origins and characteristics of Organelles ... van Dooren et al., Parasitology Today 16, 421 (2000): Tuesday April 11, 2006 ... – PowerPoint PPT presentation

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Title: Traffic to and Function of Organelles


1
  • Traffic to and Function of Organelles
  • A. Origins and characteristics of Organelles
  • Overview
  • B. Mitochondria Chloroplasts
  • Origins and characteristics
  • Structure and function of Mitochondria
  • Structure and function of Chloroplasts
  • C. Peroxisomes
  • Origins and characteristics
  • Structure and function
  • D. Apicoplasts
  • Origins and characteristics
  • Structure and function
  • E. Principles of Trafficking into Organelles
  • F. Trafficking into Mitochondria
  • G. Trafficking into Chloroplast
  • H. Peroxisomal import
  • I. Apicoplast trafficking
  • J. Comparison of trafficking in organelles

2
  • Trafficking to Organelles
  • A. Origins and characteristics of Organelles
    Overview
  • 1. Organelles in all eukaryotes
  • Nucleus
  • ER
  • Golgi
  • Lysosomes
  • Endosomes
  • Vesicles
  • PM
  • Mitochondria
  • 2. Organelles in selected eukaryotes
  • Plastids
  • Choloroplasts in plants
  • Apicoplasts in toxoplasma and plasmodium
    (apicomplexans)
  • Other secretory organelles micronemes,
    rhoptries, dense granules in apicomplexans
    (see p. 25)

3
Trafficking to Organelles A. Origins and
characteristics of Organelles Overview 3.
Evolution of Organelles
From Dyall et al. Science 304 253, 2004
4
  • Trafficking to Organelles
  • B. Mitochondria (Mt) and Chloroplasts (Ch)
  • 1. Origins and characteristics
  • a. Mt Ch are organelles enclosed within a
    double membrane
  • b. Contain their own genomes
  • c. Arose symbiotically via engulfment of
    bacteria by ancestral eukaryotic cell (primary
    endosymbiosis).

5
  • Trafficking to Organelles
  • B. Mitochondria (Mt) and Chloroplasts (Ch)
  • 1. Origins and characteristics, cont
  • d. Most of their proteins are encoded in
    the nucleus (transfer of genetic
    responsibility to the host), translated free
    in the cytosol, imported post- translationally
    in an unfolded state into Mt via specific
    targeting signals
  • e. Some of their proteins are encoded by
    DNA in the organelle
  • f. New Mt and Ch are formed by fission
    cannot be produced de novo
  • g. Contain ribosomes
  • h. Transcription and translation occur in
    matrix
  • I. N-formyl methionine as initiation codon
    just like in bacteria

6
  • Trafficking to Organelles
  • B. Mitochondria (Mt) and Chloroplasts (Ch)
  • Origins and characteristics, cont.
  • How did genes get transferred from the
    endosymbiont to the nucleus?

From Dyall et al. Science 304 253, 2004
7
  • Trafficking to Organelles
  • B. Mitochondria (Mt) and Chloroplasts (Ch)
  • 2. Mitochondrial Structure and Function
  • A. Structure Outer membrane (OM )Inner
    membrane (IM) has folds (cristae Intermembrane
    space between IM and OM Matrix is the interior.
  • B. Function by compartment
  • 1. Matrix
  • Contains mitochondrial genome
  • Encodes13 proteins (using a different genetic
    code), 2 rRNAs, 22 tRNAs
  • Contains enzymes responsible for oxidative
    metabolism
  • Oxidative metabolism Conversion of glucose to
    pyruvate (glycolysis anaerobic metabolism)
    occurs in cytosol Pyruvate fatty acids
    transported into Mt where they are converted to
    acetyl CoA, oxidized to CO2 (citric acid cycle)
    to yield ATP, NADH, and FADH2 (aerobic
    metabolism).

8
  • Trafficking to Organelles
  • B. Mitochondria (Mt) and Chloroplasts (Ch)
  • 2. Mitochondrial Structure and Function
  • B. Function by compartment
  • 1. Matrix, cont.

The Citric Acid Cycle
Fatty Acid Metabolism
9
  • Trafficking to Organelles
  • B. Mitochondria (Mt) and Chloroplasts (Ch)
  • 2. Mitochondrial Structure and Function
  • B. Function by compartment
  • 2. Membranes
  • IM NADH and FADH2 converted to ATP by
    oxidative phosphorylation energy is stored in
    proton gradient in membrane impermeable to small
    ions and molecules.
  • OM Freely permeable to small molecules (lt6kD)
    via porins that form channels

Functions in Compartments
10
  • Trafficking to Organelles
  • B. Mitochondria (Mt) Chloroplasts (Ch)
  • 2. Mt Structure and Function
  • B. Function by compartment, cont.
  • Mitochondrial proteins include proteins encoded
    in the nucleus and synthesized in the cytosol, as
    well as proteins encoded in the mitochondrion and
    synthesized in the mitochondrion.

11
  • Trafficking to Organelles
  • B. Mitochondria (Mt) Chloroplasts (Ch)
  • 3. Chloroplast Structure and Function
  • A. Structure
  • OM, IM, and intermembrane space, and stroma
    (interior space), analogous to Mt
  • Unlike Mt, Ch have an additional compartment
    (3rd membrane), the thylakoid.
  • B. Functions
  • 1. Generation of ATP.
  • 2. Photosynthetic conversion of CO2 to
    carbohydrates with production of O2.
  • 3. Synthesis of amino acids, fatty acids,
    lipid components of their own membranes.
  • 4. Reduction of nitrate to ammonia.
  • 5. Contains the Ch genome which encodes 120
    genes numerous rRNAs and tRNAs.

12
  • Trafficking to Organelles
  • Peroxisomes
  • 1. Origins and characteristics
  • 2. Peroxisomes (P) are present in all
    eukaryotic cells, and
  • a. Differ from Mt because they are surrounded by
    only a single membrane, do not contain DNA or
    ribosomes, acquire all their proteins by
    selective import from the cytosol
  • b. Post-translational mechanism of protein
    import like that of the nucleus
  • Does not involve unfolding of the cargo
  • Involves a soluble receptor in the cytosol
    that recognizes a targeting signal
  • Involves docking to proteins on the cytosolic
    surface of the peroxisome
  • c. Resemble the ER a single-membrane
    organelle replicating by fission
  • d. Likely represent a vestige of an ancient
    organelle that performed all the oxygen
    metabolism of the primitive eukaryotic cell.
    Probably served to lower oxygen which was toxic
    to the primitive cell. Later, mitochondria
    developed and rendered peroxisomes somewhat
    obsolete because they carried out the same
    reactions but now coupled to ATP formation.

13
  • Trafficking to Organelles
  • Peroxisomes
  • 2. Structure and Function
  • A. Structure Organelle surrounded by a
    single membrane.
  • B. Function (in animal cells) Contain
    peroxidases, which remove hydrogen ions from
    organic compounds, generating H2O2 (hydrogen
    peroxide).
  • RH2 O2 R H2O2
  • Contain catalases, which use H2O2 to oxidize
    other substrates, including EtOH.
  • H2O2 RH2 R 2H20
  • Oxidizes fatty acids, 2 carbons at a time, to
    acetyl CoA (occurs in mammalian Mt also).
  • Formation of specific phospholipids found in
    myelin.

14
  • Trafficking to Organelles
  • D. Apicoplasts
  • 2. Origin and Characteristics
  • Apicoplasts (Ap) are homologues of
    chloroplasts, present in Apicomplexans
    (Plasmodium, Toxoplasma, Cryptosporidium)
  • a. Complex plastids.
  • b. Differ from Mt and Ch because
  • 1. Are surrounded by four membranes.
  • 2. They originated from secondary
    endosymbiosis primitive eukaryotic ancestor
    cell engulfed another eukaryote (green alga) that
    already possessed a chloroplast.
  • 3. Contain proteins that traffic to the
    apicoplast via the secretory pathway.
  • 4. Apicoplast proteins require an ER signal
    sequence.
  • c. Resemble Mt and Ch because they
  • 1. Have their own genome (35 kB).
  • 2. Require a transit peptide signal for
    protein import.
  • d. Apicoplasts are required for infectivity.
  • e. May be excellent drug targets because they
    contain prokaryotic metabolic pathways reflecting
    their origins.

15
  • Trafficking to Organelles
  • D. Apicoplasts
  • 2. Structure and Function
  • a. Structure
  • Organelle surrounded by 4 membranes.
  • b. Function
  • Only discovered in the 1990's, so they have
    not yet been well studied Similar complex
    plastids found in algae as well
  • Dont perform photosynthesis (no genes for
    this), despite plastid origin.
  • May play other metabolic roles, i.e. AA FA
    biosynthesis, starch storage

Apicoplast (A) in Plasmodium within an infected
erythrocyte From van Dooren et al., Parasitology
Today 16, 421 (2000).
16
  • Trafficking to Organelles
  • E. Principles of Trafficking into Organelles
  • 1. Traffic into Mt, Ch, and Pe constitute
    separate trafficking routes in the cell
  • A. ER-Golgi-Lysosomes/PM
  • B. Cytoplasm-Nucleus
  • C. Cytoplasm-PM
  • D. Cytoplasm-Mt (or Ch)
  • E. Cytoplasm-Pe
  • 2. Distinguish between
  • Co-translational translocation -- ER
  • Post-translational translocation of
  • folded proteins -- nucleus
  • Post-translational translocation of unfolded
    proteins -- mitochondria
  • 3. Distinguish between
  • Transmembrane transport channel closed when not
    translocating -- ER, mitochondria, etc.
  • Gated transport diffusion vs. selective
    transport across an open pore -- nucleus
  • Vesicular transport -- Golgi, lysosomes,
    endosomes, PM.

17
  • Trafficking to Organelles
  • E. Principles of Trafficking into Organelles
  • Translocation of nuclear-encoded proteins into Mt
    Ch is typically post-translational.
  • A. Note that a very similar post-translational
    mechanism can be used in the ER of yeast and at
    bacterial plasma membranes.

18
  • Trafficking to Organelles
  • E. Principles of Trafficking into Organelles
  • 4. Translocation of nuclear-encoded proteins
    into Mt Ch is typically post-translational.
  • In contrast to the co-translational
    trans-location that in the eukaryotic ER,
    post-translational translocation into MT/Ch
    requires
  • 1. that newly-synthesized Mt protein be
    kept unfolded before translocation
  • 2. the presence of a Mt signal sequence
    (also called presequence) that directs the chain
    to the OM
  • 3. protein translocators in organelle mb
    that allow translocation across membrane
  • 4. proteins targeted to organelles with
    multiple membranes often encode a second signal
    (transit peptide) to allow transport across inner
    membrane

Translocators in Mitochondrial Membrane
19
  • Trafficking to Organelles
  • F. Mitochondrial import
  • 1. Signals and Translocators
  • a. Mt import signal (pre-sequence) is an
    amphipathic helix

20
  • Trafficking to Organelles
  • F. Mitochondrial import
  • 1. Signals and Translocators, cont.
  • b. Presequence binds to receptor on Mt
    surface.
  • c. Insertion into the TOM complex
    translocator across the outer Mt mb. Used by all
    proteins imported into Mt mediated by the
    presequence.
  • d. Insertion into TIM complexes (22 23)
    translocators across inner Mt mb. mediated by
    a second sorting signal located distal to the
    presequence and, in the case of transmembrane
    proteins, a stop-transfer signal.
  • e. Presequence removed in the matrix (or
    the intermembrane space) by signal peptidase.
  • f. Thus, Mt proteins cross both membranes,
    which become closely apposed, at once rather than
    one at a time.

21
  • Trafficking to Organelles
  • F. Mitochondrial import
  • 1. Signals and Translocators, cont.
  • g. OxA complex mediates insertion of proteins
    synthesized in Mt into IM.
  • h. Also proteins that are to be inserted into
    the IM are sometimes first translocated into the
    matrix, have their pre-sequence cleaved, then
    the 2nd signal acts as an N-terminal signal
    directing them to be re-inserted into the IM via
    the OxA complex, with a stop-transfer to hold
    them in a transmembrane orientation.

22
  • Trafficking to Organelles
  • F. Mitochondrial import, cont.
  • 2. Chaperones act on both sides of the
    mitochondrial membrane during translocation
  • a. Hsp70 maintains newly-synthesized Mt
    protein in cytosol in unfolded state.
  • Release of protein from Hsp70 requires ATP
    hydrolysis.
  • b. Translocation through the TIM complex
    requires electrochemical H gradient
  • maintained by pumping H ions from matrix to
    inter Mt membrane space, driven by electron
    transport in inner mitochondrial membrane.
  • Thus, electron transport in inner Mt membrane
    not only is the source of most of the cells ATP,
    but also transport of Mt proteins through TIM
    complex.

23
  • Trafficking to Organelles
  • F. Mitochondrial import
  • 2. Chaperones act on both sides of the Mt
    membrane during translocation, cont.
  • c. Another Hsp 70 is associated with the TIM
    complex and acts as a motor that drives import.
  • d. The translocated Mt protein is then
    transferred to an Hsp 60 chaperone in the matrix,
    which promotes Mt protein folding (and also
    hydrolyzes ATP).

Two different Models for how Mt hsp70 drives
protein import into the Mt
24
  • Trafficking to Organelles
  • G. Chloroplast import is analogous to Mt import,
    except
  • 1. GTP and ATP are used for energy at OM and
    IM.
  • 2. Electrochemical gradient is present at the
    thylakoid membrane.
  • 3. Translocation complex in OM is Toc
    translocation complex in IM is Tic.
  • 4. Transit peptide directs translocation across
    OM and IM, and is removed by cleavage in the
    stroma, exposing in some cases a second signal
    sequence which directs transport across the
    thylakoid membrane.
  • 5. While the signal sequences for Mt and Ch
    resemble each other, since both occur in plant
    cells, they need to be different enough to direct
    specific targeting to the right compartment.

25
  • Trafficking to Organelles
  • H. Peroxisomal import
  • 1. Uses a 3 aa signal (Ser-Lys-Leu).
  • 2. Attachment of this signal on a cytosolic
    protein results in peroxisomal import.
  • 3. Driven by ATP hydrolysis.
  • 4. Peroxins are proteins that participate in
    peroxisomal import.
  • 5. Unlike in the case of mitochondria or
    chloroplasts, peroxisomal proteins do not have to
    be unfolded to be transported.
  • 6. A soluble import receptor binds the cargo
    in the cytosol and accompanies it into the
    peroxisomes. After cargo releases, the receptor
    cycles back to the cytosol. This implies that an
    export system exists, but this has yet to be
    found.

26
  • Trafficking to Organelles
  • I. Apicoplast import
  • 1. Related to chloroplasts but surrounded by 4
    mbs.
  • 2. Evidence exists for a classical secretory
    system in apicoplasts.
  • 3. However, additional organelles exist
    (micronemes, rhoptries, and dense granules, and
    PVM). Also BFA not effective.
  • 4. Leader sequence contains signal peptide (SP)
    transit peptide (TP). SP targets proteins to
    secretory system SP TP targets to apicoplast.
  • 5. Toxo and plasmodium leader sequences function
    interchangeably. Chloroplast TPs from plants can
    also substitute for apicoplast TP.
  • 6. TIC and TOC homologues are in apicoplasts.
  • 7. Unclear if apicoplast is proximal or distal to
    Golgi.

Legend (a) Translation of protein with signal
peptide followed by (b) Co-translational
insertion into first membrane via SP Second
membrane recognizes TP (c) Another Toc complex
may be present in final set of membranes, perhaps
acting along with a Tic complex (d).
From van Dooren et al., Parasit. Today 16, 421
(2000)
27
  • Trafficking to Organelles
  • I. Apicoplast Import
  • Apicoplast targeting is only one of the
    trafficking complexities of Toxo

From Joiner and Roos, J. Cell Biol. 157 557-563,
2002
28
  • Trafficking to Organelles
  • I. Apicoplast Import
  • Apicoplast targeting is only one of the
    trafficking complexities of Plasmodium

From van Dooren et al., Parasitology Today 16,
421 (2000)
29
Additional Reading (not required) Dyall SD,
Brown MT, Johnson PJ. Ancient invasions from
endosymbionts to organelles.Science. 2004 Apr
9304(5668)253-7. Review. Osteryoung KW,
Nunnari J. The division of endosymbiotic
organelles. Science. 2003 Dec 5302(5651)1698-704
. Review. Wiedemann N, Pfanner N, Chacinska A.
Chaperoning through the mitochondrial
intermembrane space.Mol Cell. 2006 Jan
2021(2)145-8. Review. Wickner W, Schekman R.
Protein translocation across biological
membranes.Science. 2005 Dec 2310(5753)1452-6.
Review. Horrocks P, Muhia D. Pexel/VTS a
protein-export motif in erythrocytes infected
with malaria parasites. Trends Parasitol. 2005
Sep21(9)396-9. van Dooren GG, Waller RF,
Joiner KA, Roos DS, McFadden GI. Traffic jams
protein transport in Plasmodium
falciparum.Parasitol Today. 2000
Oct16(10)421-7. Review.
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