Protein Sorting and Transport

8/2/2019 Protein Sorting and Transport

1/17

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


2/17

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


3/17

Experimental Systems

1. Autoradiography, Pulse-Chase, and GreenFluorescent Protein Experiments

2. Differential Staining

3. Cell Fractionation

4. Genetic Mutant Analysis


4/17

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


5/17

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


6/17

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


7/17


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


8/17


ProteinTransport

Transport of chloroplast proteins is similar tomitochondrial proteins

Thylakoid proteins require an extra thylakoid

transport sequence & import proteins

O f C


9/17

Overview of the Cytomembrane

System

1. Endoplasmic Reticulum

2. Golgi Apparatus

3. Intercompartmental Transport Vesicles

4. Secretory Vesicles

5. Endocytotic Vesicles

6. Lysosomes


10/17

Endoplasmic Reticulum

1. Functions of the Smooth ER

Steroid Hormone Synthesis

Detoxification in Liver

Release of glucose from liver

Sequestering calcium ions


11/17

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


12/17

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


13/17

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


14/17

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.


15/17

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.


16/17


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.


17/17


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

Documents

Protein Sorting and Transport