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.