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Protein Sorting & Transport
Paths of Protein TraffickingNuclear Protein Transport
Mitochondrial & Chloroplast Transport
Experimental SystemsOverview of the Cytomembrane System
The Endoplasmic Reticulum
The Golgi ApparatusVesicular Transport between Compartments
Exocytosis
Endocytosis and Lysosomes
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Paths of Protein Trafficking
Completed protein
via Posttranslational ImportGoes to:
Cytosol, Nucleus, Mitochondria, Plastids, Peroxisomes
Plasma Membrane
Secretory Vesicles
Late Endosomes mature
to form Lysosomes
Vesicles from Golgi
fuse with Early Endosomes
to form Late Endosomes
Golgi Apparatusvia Vesicular Transport
Protein is further modifiedand goes to:
Ribosomes with partially synthesized proteins:
Attach to Endoplasmic ReticulumProtein is imported into ER via Cotranslational Import
Protein is modified in ER and goes to:
Ribosomes in Cytosol
NucleusmRNA producedby transcription
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Experimental Systems
1. Autoradiography, Pulse-Chase, and GreenFluorescent Protein Experiments
2. Differential Staining
3. Cell Fractionation
4. Genetic Mutant Analysis
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Nuclear Protein Transport
Nuclear Pores ~9 nm formed from nucleoporinproteins
Smaller proteins (~17kD or less) may diffuse
freely; larger proteins must be imported bygated pore mechanism
Proteins targeted for nucleus have nuclear
localization signals
Most NLS must be recognized & bound by
nuclear import receptors or nuclear export
receptors
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Nuclear Protein TransportDuring nuclear protein transport, much of the
tertiary structure of the transported proteinremains intact, due to large size of nuclear pores
Energy is provided by GTP hydrolysis via Ran, a
GTPase found both in cytoplasm & nucleusIn the cytoplasm a GTPase activating factor
(GAP) triggers hydrolysis of GTP by Ran; in the
nucleus a GDP-GTP exchange factor (GEF)The gradient of Ran-GDP and Ran-GTP across
the nuclear membrane drives transport in the
appropriate direction
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Mitochondrial/Chloroplast
ProteinTransport
Mitochondrial proteins that are encoded by nucleargenes possess mitochondrial import sequences
Transport from the cytosol into the mitochondria ismediated by a TOM complex and two TIM complexes
The SAM complex mediates proper folding of outermembrane proteins with -barrel structures
Another complex (OXA) mediates insertion ofmitochondrial-encoded proteins into inner membrane &also contributes to insertion of some nuclear-encodedproteins
Proteins must be unfolded, either by chaperones or by
specialized unfolding proteins
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Mitochondrial/Chloroplast
ProteinTransport
ATP hydrolysis & a H+ gradient drive proteintransport into mitochondria via a ratcheting
mechanism
Integral proteins possess stop transportsequences that interrupt the transport process to
create the transmembrane domains; formation of
the transmembrane domain may be eithermediated by TIM23, TIM22 (specialized for
multipass proteins), or OXA
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Mitochondrial/Chloroplast
ProteinTransport
Transport of chloroplast proteins is similar tomitochondrial proteins
Thylakoid proteins require an extra thylakoid
transport sequence & import proteins
O f C
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Overview of the Cytomembrane
System
1. Endoplasmic Reticulum
2. Golgi Apparatus
3. Intercompartmental Transport Vesicles
4. Secretory Vesicles
5. Endocytotic Vesicles
6. Lysosomes
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Endoplasmic Reticulum
1. Functions of the Smooth ER
Steroid Hormone Synthesis
Detoxification in Liver
Release of glucose from liver
Sequestering calcium ions
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Endoplasmic Reticulum
2. Functions of the Rough ER
Cotranslational import of proteins destined for the
ER-Golgi pathway
Synthesis of membrane lipids and generation of lipidcompositional asymmetry
Protein glycosylation: Synthesis of the core portion
of an N-linked oligosaccharide
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Golgi Apparatus
Modification of N-linked oligosaccharides
Synthesis of O-linked oligosaccharides
Phosphorylation of mannose (on N-linked
oligosaccharide) on proteins targeted for lysosomesSorting of proteins into secretory vesicles or primary
lysosomes
V i l T t b t
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Vesicular Transport between
Compartments
1. Transport vesicles are generally covered with coatproteins:
COPII-coated vesicles: move proteins from ER to cis-Golgi
COPI-coated vesicles: move proteins from cis-Golgi toER; also possibly from ER to Golgi and between Golgicisternae
Clathrin-coated vesicles: move proteins from the trans-Golgi to the plasma membrane or lysosomes
V i l T t b t
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Vesicular Transport between
Compartments
2. Receptor protein systems (SNAREs) are believed totarget and dock specific vesicles to the correct
compartment
3. At each step in the cytomembrane pathway, proteinsthat should stay in the previous compartment are
retrieved by membrane-bound receptors and sent back
to the correct compartment.
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Exocytosis
Secretory vesicles (from the trans-Golgi) are targetedto the plasma membrane, with which they fuse.
The soluble contents of the vesicles are released to the
outside, and the vesicle membrane becomes part ofthe PM.
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Endocytosis and Lysosomes
Endocytotic vesicles form from clathrin-coatedpits in the plasma membrane. This process is
often mediated by receptor proteins that bind to
specific ligands that the cell need to transport.Generally, the endocytotic vesicles fuse with
primary lysosomes (from the trans-Golgi) to form
secondary lysosomes.
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Endocytosis and Lysosomes
Lysosomes contain many different hydrolyticenzymes that process the contents of the
endosome.
Lysosome proteins are recognized and sorted bythe trans-Golgi due to the mannose 6-phosphate
residues on N-linked oligosaccharide