Transcript Lecture 5 Tues 4-11-06

Traffic to and Function of Organelles
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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
Tuesday April 11, 2006
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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)
Other unique organelles (see p. 26 )
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A. Origins and characteristics of Organelles: Overview
3. Evolution of Organelles:
From Dyall et al. Science 304: 253, 2004
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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).
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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 posttranslationally 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
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B. Mitochondria (Mt) and Chloroplasts (Ch):
1. Origins and characteristics, cont.
How did genes get transferred from the
endosymbiont to the nucleus?
From Dyall et al. Science 304: 253, 2004
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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).
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B. Mitochondria (Mt) and Chloroplasts (Ch):
2. Mitochondrial Structure and Function:
B. Function by compartment :
1. Matrix, cont.
Fatty Acid Metabolism
Tuesday April 11, 2006
The Citric Acid Cycle
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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 (<6kD) via porins that form channels
Functions in Compartments
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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.
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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.
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C.
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.
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C. 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 + R’H2 = 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.
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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.
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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
Don’t 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).
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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.
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E. Principles of Trafficking into Organelles:
4.
Translocation of nuclear-encoded proteins into Mt & Ch is typically posttranslational.
A. Note that a very similar post-translational mechanism can be used in
the ER of yeast and at bacterial plasma membranes.
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E. Principles of Trafficking into Organelles:
4. Translocation of nuclear-encoded proteins
into Mt & Ch is typically post-translational.
B.
1.
2.
3.
4.
Translocators in Mitochondrial
Membrane:
In contrast to the co-translational translocation that in the eukaryotic ER,
post-translational translocation into MT/Ch
requires:
that newly-synthesized Mt protein be kept
unfolded before translocation
the presence of a Mt signal sequence (also
called presequence) that directs the chain
to the OM
protein translocators in organelle mb that
allow translocation across membrane
proteins targeted to organelles with
multiple membranes often encode a
second signal (transit peptide) to allow
transport across inner membrane
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F. Mitochondrial import:
1. Signals and Translocators:
a. Mt import signal (pre-sequence) is an amphipathic helix
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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.
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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 Nterminal 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.
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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 cell’s ATP, but also transport of Mt proteins through TIM complex.
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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
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G. Chloroplast import is analogous to Mt import, except:
1.
2.
3.
4.
GTP and ATP are used for energy at OM and IM.
Electrochemical gradient is present at the thylakoid membrane.
Translocation complex in OM is Toc; translocation complex in IM is Tic.
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.
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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.
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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)
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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
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I. Apicoplast Import:
Apicoplast targeting is only one of the trafficking complexities of Plasmodium:
From
van Dooren et
al., Parasitology
Today 16, 421
(2000):
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Additional Reading (not required):
Dyall SD, Brown MT, Johnson PJ. Ancient invasions: from endosymbionts to
organelles.Science. 2004 Apr 9;304(5668):253-7. Review.
Osteryoung KW, Nunnari J. The division of endosymbiotic organelles. Science. 2003 Dec
5;302(5651):1698-704. Review.
Wiedemann N, Pfanner N, Chacinska A. Chaperoning through the mitochondrial
intermembrane space.Mol Cell. 2006 Jan 20;21(2):145-8. Review.
Wickner W, Schekman R. Protein translocation across biological membranes.Science. 2005
Dec 2;310(5753):1452-6. Review.
Horrocks P, Muhia D. Pexel/VTS: a protein-export motif in erythrocytes infected with malaria
parasites. Trends Parasitol. 2005 Sep;21(9):396-9.
van Dooren GG, Waller RF, Joiner KA, Roos DS, McFadden GI. Traffic jams: protein
transport in Plasmodium falciparum.Parasitol Today. 2000 Oct;16(10):421-7. Review.
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