1. Protein Sorting & Transport
Paths of Protein Trafficking
Nuclear Protein Transport
Mitochondrial & Chloroplast Transport
Experimental Systems
Overview of the Cytomembrane System
The Endoplasmic Reticulum
The Golgi Apparatus
Vesicular Transport between Compartments
Exocytosis
Endocytosis and Lysosomes

3. Experimental Systems
1. Autoradiography, “Pulse-Chase,” and Green Fluorescent Protein Experiments
2. Differential Staining
3. Cell Fractionation
4. Genetic Mutant Analysis

4. Nuclear Protein Transport
Nuclear Pores ~9 nm formed from nucleoporin proteins
Smaller proteins (~17kD or less) may diffuse freely; larger proteins must be imported by “gated 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. Nuclear Protein Transport
During nuclear protein transport, much of the tertiary structure of the transported protein remains intact, due to large size of nuclear pores
Energy is provided by GTP hydrolysis via Ran, a GTPase found both in cytoplasm & nucleus
In 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. Mitochondrial/Chloroplast ProteinTransport
Mitochondrial proteins that are encoded by nuclear genes possess mitochondrial import sequences
Transport is mediated by a TOM complex and two TIM complexes; another complex (OXA) mediates insertion of mitochondrial-encoded proteins into inner membrane & also contributes to insertion of some nuclear-encoded proteins
Proteins must be unfolded, either by chaperones or by specialized unfolding proteins

7. Mitochondrial/Chloroplast ProteinTransport
ATP hydrolysis & a H+ gradient drive protein transport into mitochondria via a ratcheting mechanism
Integral proteins possess stop transport sequences that interrupt the transport process to create the transmembrane domains; formation of the transmembrane domain may be either mediated by TIM23, TIM22 (specialized for multipass proteins), or OXA

8. Mitochondrial/Chloroplast ProteinTransport
Transport of chloroplast proteins is similar to mitochondrial proteins
Thylakoid proteins require an extra thylakoid transport sequence & import proteins

9. Overview of the Cytomembrane System
1. Endoplasmic Reticulum
2. Golgi Apparatus
3. Intercompartmental Transport Vesicles
4. Secretory Vesicles
5. Endocytotic Vesicles
6. Lysosomes

10. Endoplasmic Reticulum
1. Functions of the Smooth ER
Steroid Hormone Synthesis
Detoxification in Liver
Release of glucose from liver
Sequestering calcium ions

11. 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 lipid compositional asymmetry
Protein glycosylation: Synthesis of the core portion of an N-linked oligosaccharide

12. Golgi Apparatus Modification of N-linked oligosaccharides
Synthesis of O-linked oligosaccharides
Phosphorylation of mannose (on N-linked oligosaccharide) on proteins targeted for lysosomes
Sorting of proteins into secretory vesicles or primary lysosomes

13. Vesicular Transport between Compartments
1. Transport vesicles are generally covered with coat proteins:
COPII-coated vesicles: move proteins from ER to cis-Golgi
COPI-coated vesicles: move proteins from cis-Golgi to ER; also possibly from ER to Golgi and between Golgi cisternae
Clathrin-coated vesicles: move proteins from the trans-Golgi to the plasma membrane or lysosomes

14. Vesicular Transport between Compartments
2. Receptor protein systems (SNAREs) are believed to target and dock specific vesicles to the correct compartment
3. At each step in the cytomembrane pathway, proteins that should stay in the previous compartment are retrieved by membrane-bound receptors and sent back to the correct compartment.

15. Exocytosis
Secretory vesicles (from the trans-Golgi) are targeted to the plasma membrane, with which they fuse.
The soluble contents of the vesicles are released to the outside, and the vesicle membrane becomes part of the PM.

16. Endocytosis and Lysosomes
Endocytotic vesicles form from clathrin-coated pits 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. Endocytosis and Lysosomes
Lysosomes contain many different hydrolytic enzymes that process the contents of the endosome.
Lysosome proteins are recognized and sorted by the trans-Golgi due to the mannose 6-phosphate residues on N-linked oligosaccharide