Intracellular Compartments
and Protein Sorting
Intracellular Compartments and
Protein Sorting
►Functionally distinct membrane bound organelles
►10 billion proteins of 10,000-20,00 diff kinds
►Complex delivery system
Compartmentalization of Cells
Membranes
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Partition cell
Important cellular functions
Impermeable to most hydrophobic molecules
contain transport proteins to import and export specific molecules
Mechanism for importing and incorporating organelle specific proteins
that define major organelles
Compartmentalization of Cells
All Eucaryotic Cells Have Same Basic Set of Membrane Bound Organelles
Compartmentalization of Cells
Compartmentalization of Cells
Compartmentalization of Cells
Major Organelles
►Nucleus
►Cytosol
►ER
►Golgi Apparatus
►Mitochondria and Chloroplast
►Lysosomes
►Endosomes
►Peroxisomes
Compartmentalization of Cells
►Occupy 50% cell volume
►Perform same basic function
►Vary in size and abundance
►May take on additional functions
►Position dictated by cytoskeleton
Compartmentalization of Cells
Topology governed by evolutionary origins
Invagination of pm creates organelles such as nucleus that are
topologically equivalent to cytosol and communicate via pores
Compartmentalization of Cells
Topology governed by evolutionary origins
Endosymbiosis of mito and plastids creates double
membrane organelle (have own genome)
Compartmentalization of Cells
Topology governed by evolutionary origins
Organelles arising from pinching off of pm have interior
equivalent to exterior of cell
Compartmentalization of Cells
3 Types of Transport Mechanisms
1. Gated Transport:
gated channels
topologically equivalent spaces
2. Transmembrane Transport:
protein translocators
topologically distinct space
3. Vesicular transport:
membrane enclosed intermediates
topologically equivalent spaces
Compartmentaliztion of Cells
Families of Intracellular Compartments:
1. nucleus and cytosol
2. organelles in the secretory pathway
3. mitochondria
4. plastid
Transport guided by:
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2.
sorting signals in transported proteins
complementary receptor proteins
Compartmentalization of Cells
2 Types of Sorting Signals in Proteins
1.
Signal Sequence
continuous sequence of 15-60 aa
sometimes removed from finished protein
sometimes a part of finished protein
2.
Signal Patch
specific 3d arrangement of atoms on protein surface; aa’s distant
persist in finished protein
Compartmentalization of Cells
Compartmentalization of Cells
Signal Sequences/Patches
Direct Proteins to Final Destination
Signal patches direct proteins to:
1. nucleus
2. lysosomes
Signal Sequences direct proteins to:
1. ER proteins possess N-terminal signal of 5-10 hydrophobic aa
2. mito proteins have alternating + chg aa w/ hydrophobic aa
3. proxisomal proteins have 3 aa at C-terminus
Compartmentalization of Cells
Sorting signals recognize complementary sorting receptors
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Receptors unload cargo
Function catalytically and are reusable
Compartmentalization of Cells
Organelles Cannot be Constructed Denovo
► Organelles reproduced via binary fission
► Organelle cannot be reconstructed from DNA alone
► Info in form of one protein that pre-exists in organelle mem is required
and passed on from parent to progeny
► Epigenetic information essential for propogation of cell’s compartmental
organization
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Envelope
►Two concentric membranes
-Outer membrane contiguous w/ER
-Inner membrane contains proteins that
act as
binding sites for chromatin and nuclear
lamina
►Perforated by nuclear pores for selective
import and export
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Pore Complex
►mass of 125 million; ~50 different
proteins arranged in octagon
►Typical mammalian cell 3,000-4,000
►Contains >1 aqueous channels thru
which sm molec can readily pass
<5,000; molec > 60,000 cannot pass
►Functions ~diaphram
►Receptor proteins actively transport
molec thru nuclear pore
Transport of Molecules Btwn Nucleus and Cytosol
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Localization Signal
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Generally comprised of two short sequences rich in + chged aa lys & arg
Can be located anywhere
Thought to form loops or patches on protein surface
Resident, not cleaved
Transport thru lg aqueous pores as opposed to translocator proteins
Transports proteins in folded state
Energy requiring process
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Import- the players
► Importins = cytosolic receptor protein binds to NLS of “cargo” proteins
► Nucelar Export Receptors = binds macromolecules to be exported from nucelus
► Adaptors = sometimes required to bind target protein to nuclear receptor
► Ran = cytosolic GTP/GDP binding protein complexes with importins in the cytosol.
► Fibril proteins and nucleoporins contain phenylalanine/glycine repeats (FG) repeats.
Repeats transiently bound and released by importin/cargo/Ran-GDP, causing the
complex to “hop” into the nucleus
Import Receptors release cargo in nucleus and return to cytosol
Export Receptors release cargo in cytoplasm and return to nucleus
Transport of Molecules Btwn Nucleus and Cytosol
Ran GTPase= molecular switch
► Drives directional transport in appropriate directin
► Conversion btwn GTP and GDP bound states mediated by Ran specific regulatory proteins
GAP converts RNA-GTP to Ran-GDP via GTP hydrolysis
GEF promotes exchg of GDP for GTP converting Ran-GDP to Ran-GTP
► Ran GAP in cytosol thus more Ran-GDP in cytosol
► Ran GEF in nucleus thus more Ran-GTP in nucleus
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Export
► Works like import in reverse
► Export receptors bind export signals and nucleoporins to guide
cargo thru pore
► Import and export receptors member of same gene family
Transport of Molecules Btwn Nucleus and Cytosol
Regulation Afforded by Access to Transport Machinery
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Controlling rates of import and export determines steady state location
► phosphorylation/dephosphorylation of adjacent aa may be required for receptor binding
► Cytosolic anchor or mask proteins block interaction w/ receptors
► Protein made and stored in inactive form as ER transmembrane protein
Transport of Molecules Btwn Nucleus and Cytosol
Control of mRNA Export
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Proteins w/ export signals loaded onto RNA during transcription and
processing (RNP)
Export signals guide RNA out of nucleus thru pores via exportin proteins than
bind RNP
Export mediated by transient binding to FG repeats
Imature mRNAs retained by anchoring to transcription and splicing machinery
Proteins disassociate in cytosol and return to nucleus
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Lamina
►Meshwork of intermediate filaments
►Maintenance of nuclear shape
►Spacial organization of nuclear pores
►Regulation of transcription
►Anchoring of interphase chromatin
►DNA replication
►Phosphorylation causes depolymerizes
during mitosis when nucleus disassembles
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear envelop disassembles during mitosis and reassembles
when ER wraps around chromosomes and begins to Fuse
Protein Transport into the
Mitochondria and Chloroplast
Subcompartments of the Mitochondria and Chloroplast
Protein Transport into the
Mitochondria and Chloroplast
Translocation into Mitochondrial Matrix Governed by:
1.
Signal Sequence (amphipathic alpha helix cleaved after import)
2.
Protein Translocators
Protein Transport into the
Mitochondria and Chloroplast
Players in Protein Translocation of Proteins in Mitochondria
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TOM- functions across outer membrane
TIM- functions across inner membrane
OXA- mediates insertion of IM proteins syn w/in mito and helps to
insert proteins initially transported into matrix
Complexes contain components that act as receptors and
others that form translocation channels
Protein Transport into the
Mitochondria and Chloroplast
Import of Mitochondrial Proteins
►Post-translational
►Unfolded polypeptide chain
1.
precursor proteins bind to receptor proteins of TOM
2.
interacting proteins removed and unfolded polypetide is fed into
translocation channel
►Occurs contact sites joining IM and OM
TOM transports mito targeting signal across OM and once it reaches IM
targeting signal binds to TIM, opening channel complex thru which protein
enters matrix or inserts into IM
Protein Transport into the
Mitochondria and Chloroplast
Import of Mitochondrial Proteins
►Requires energy in form of ATP and H+ gradient and assitance of hsp70
-release of unfolded proteins from hsp70 requires ATP hydrolysis
-once thru TOM and bound to TIM, translocation thru TIM requires
electrochemical gradient
Protein Transport into the
Mitochondria and Chloroplast
Protein Transport into IM or IM Space Requires 2 Signal Sequences
1. Second signal =hydrophobic sequence; immediately after 1st signal sequence
2. Cleavage of N-terminal sequence unmasks 2nd signal used to translocate protein
from matrix into or across IM using OXA
3. OXA also used to transport proteins encoded in mito into IM
4. Alternative route bypasses matrix; hydrophobic signal sequence = “stop
transfer”
Protein Transport into the
Mitochondria and Chloroplast
Protein Transport into Chloro Similar to Transport into Mito
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occur posttranslationally
Use separate translocation complexes in ea membrane
Translocation occurs at contact sites
Requires energy and electrochemical gradient
Use amphilpathic N-terminal signal seq that is removed
Like the mito a second signal sequence required for translocation
into thylakoid mem or space
Protein Transport into the
Mitochondria and Chloroplast
Peroxisomes and Protein Import
Peroxisomes
►Use O2 and H2O2 to carry out oxidative rxns
►Remove H from specific organic compounds RH2 + O2
R + H2O2
►Catalases use H2O2 to oxidize other substances, particularly in liver and kidney detoxification
H2O2 + R’H2
R’ + H2O
►Beta Oxidation
►Formation of plasmalogens (abundant class of phospholipids in myelin)
►Photorespiration and glyoxylate cycle in plants
Peroxisomes and Protein Import
Peroximsomes in Plants
►Site of Photorespiration= glycolate pathway in leaves
►Called glyoxysomes in seeds where fats converted into
sugar
Proxisomes and Protein Import
►Peroxisomes arise from pre-existing peroxisomes
►Signal sequence of 3 aa at COOH end of peroxisomal proteins= import signal
►Some have signal sequence at N-terminus
►Involves >23 distinct proteins
►Driven by ATP hydrolysis
►Import mechanism distinct, not fully characterized
►Oligomeric proteins do not unfold when imported
►Zellweger Disease= peroxisomal deficiency
ER and Protein Trafficking
Endoplasmic Reticulum
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Occupies >= 50% of cell volume
Continuous with nuclear membrane
Central to biosyn macromolecules used to construct other organelles
Trafficking of proteins to ER lumen, Gogli, lysosome or those to be secreted
from cell
ER and Protein Trafficking
ER Central to Protein Synthesis and Trafficking Removes 2 Types of Proteins from Cytosol:
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transmembrane proteins partly translocated across ER embedded in it
2.
water soluble proteins translocated into lumen
ER and Protein Trafficking
Quantity of SER and ER Dependent Upon Cell Type
RER assoc. w/ protein synthesis
SER assoc. lipid biosynthesis, detoxification, steroid synthesis, Ca2+ storage
ER and Protein Trafficking
Import of Proteins into ER
►Occurs co-translationally
►Signal recognition sequence recognized by SRP
►SRP recognized by SRP receptor
►Protein Translocator
ER and Protein Trafficking
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Hydrophobic signal sequence of diff sequence and shape
SRP lg hydrophobic pocket lined by Met having unbranched flexible
side chains
Binding of SRP causes pause in protein synthesis allowing time for
SRP-ribosome complex to bind to SRP receptor
ER and Protein Trafficking
Protein to be imported passes through an aqueous pore in
the translocator that is a dynamic structure
►Sec61 protein translocator
►Signal sequence triggers opening of pore
►Translocator pore closes when ribo not present
ER and Protein Trafficking
Some proteins are imported in to ER by a posttranslational mechanism
►Proteins released into cytoplasm
►Binding of chaperone proteins prevents them from folding
►Translocation occurs w/out ribo sealing pore
►Mechanism whereby protein moves through pore unkwn
ER and Protein Trafficking
Signal Sequence is Removed from Soluble Proteins
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Two signaling functions:
1) directs protein to ER membrane
2) serves as “start transfer signal” to open pore
Signal peptidase removes terminal ER signal sequence upon
release of protein into the lumen
ER and Protein Trafficking
Single Pass Transmembrane Proteins
1.
N-terminal signal sequence initiates translocation and additional hydrophobic “stop
sequence anchors protein in membrane
2.
Signal sequence is internal and remains in
lipid bilayer after release from translocator
3.
Internal signal sequence in opposite
orientation
4.
Orientation of start-transfer sequence
governed by distribution of nearby chg aa
ER and Protein Trafficking
Multipass Transmembrane Proteins
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Combinations of start- and stop-transfer signals determine topology
Whether hydrophobic signal sequence is a start- or stop-transfer
sequence depends upon its location in polypeptide chain
All copies of same polypeptide have same orientation
ER and Protein Trafficking
Folding of ER Resident Proteins
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ER resident proteins contain an ER
retention signal of 4 specific aa at Cterminus
PDI protein disulfide isomerase oxidizes free
SH grps on cysteines to from disulfide
bonds S-S allowing proteins to refold
BiP chaperone proteins, pulls proteins
posttranslationally into ER thru translocator
and assists w/ protein folding
ER and Protein Trafficking
Glycolsylation of ER Proteins
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Most soluble and transmembrane proteins made in ER are
glycolsylated by addition of an oligosaccharide to Asn
Precursor oligosaccharide linked to dolichol lipid in ER mem, in
high energy state
Transfer by oligosaccharyl transferase occurs almost as soon as
polypeptide enters lumen
ER and Protein Trafficking
Oligosaccharide assembled sugar by sugar onto carrier lipid dolichol
ER and Protein Trafficking
Retrotranslocation
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Improperly folded ER proteins are exported and degraded in cytosol
Misfolded proteins in ER activate an “Unfolded Protein Response” to
increase transcription of ER chaperones and degradative enzymes
ER and Protein Trafficking
The Unfolded Protein Response
ER and Protein Trafficking
Assembly of Lipid Bilayers on ER
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ER synthesizes nearly all major classes of lipids
Phospholipid synthesis occurs on cytoplasmic face by enzymes in
mem
Acyl transferases add two FA to glycerol phosphate producing
phosphatidic acid
Later steps determine head group
ER and Protein Trafficking
Assembly of Lipid Bilayers on ER
►Scramblase phospholipid translocator equilibrates phospholipids
distribution
►Flipasses of PM responsible for asymmetric distribution of phospholipids
ER and Protein Trafficking
Phospholipid Exchange Proteins
►Transfer individual phosphlipids between membranes at random btwn
all membranes
►Exchange protein specificity
►Extracts phospholipid and diffuses away w/ it buried w/in lipid binding
site; discharges phospholipid when it encounters another membrane
Transport of Molecules Btwn Nucleus and Cytosol