Labs 5 - 8 Protein Trafficking

1. Labs # 5 - # 8Protein Trafficking There and Back Again

2. Eukaryotes and Compartmentalization Eukaryotic cells have many internal compartments/organelles. What gives these compartments their functional identity? How is the functional identity of an organelle maintained? Complex questions like these must be approached from multiple directions in order to arrive at meaningful conclusions. Cells invest a great deal of energy in maintaining compartments.Cells invest a great deal of energy in maintaining compartments.

4. Protein What do you know about protein? Sizes inkDa or kilodaltons Dalton is the mass of hydrogen molecule = 1.66 x 10 -24 grams Average aa = 110 daltonsSizes inkDa or kilodaltons Dalton is the mass of hydrogen molecule = 1.66 x 10 -24 grams Average aa = 110 daltons

5. Why does pH matter? The cytosol of a human cell is about 7.4 (lysosome is about 4). Proteins are sensitive to pH. As the pH drops, the net charge on a given protein molecule becomes more positive. As the pH rises the net charge becomes more negative. Many of the opportunities that the ionizable R groups have for interactions are altered away from optimal. This can change protein conformation as well as binding affinities for substrates and cofactors.

6. Where does protein come from?

7. Where are proteins made? What proteins are translated in the cytoplasm? Possible fates? What proteins are translated on ER associated ribosomes? Possible fates?What proteins are translated in the cytoplasm? Possible fates? What proteins are translated on ER associated ribosomes? Possible fates?

8. The ER is the first stop for all proteins destined for: Incorporation into the ER itself Incorporation into the Golgi Later secretory pathway organelles Plasma membrane Secretion from cell The default pathway takes these proteins either to the cell membrane or secretes them from the cell. Some exceptions exist. A few secreted proteins are targeted out of the cell from the cytoplasm! How are proteins diverted from this pathway? Some exceptions exist. A few secreted proteins are targeted out of the cell from the cytoplasm! How are proteins diverted from this pathway?

9. We will focus on lysome/vacuole targeting How are proteins diverted from the default pathway and sent to lysosomes (in mammals) and vacuoles (in yeast)? Lysosomes and vacuoles are equivalent compartments.

10. Endosomes Segregate endocytosed macromolecules. So endosomes serve as sites of organization. First destination of newly synthesized lysosomal/vacuolar hydrolases arriving fresh from the TGN. Sorting site for PM receptors and other PM proteins internalized from the cell surface. Major traffic hub. The endosomal system is made up of multiple vesicular compartments: early endosome and late endosome/MVB.The endosomal system is made up of multiple vesicular compartments: early endosome and late endosome/MVB.

11. Endocytosis Internalized molecules enter early sorting endosomes. Molecules are segregated to tubular or central portions of the early sorting endosome. Central portion matures into MVB/late endosome by involution of the membrane.

13. What choices are available to an soluble endocytosed macromolecule? Recycle back to the Cell Surface. Recycle back to TGN Or���������

14. ��.DOOM! Degradation of macromolecules in the lysosome/vacuole. 

16. How Complex is the System? The proteins and lipids synthesized in the ER provide the foundation for assembly and function of all compartments comprising the exocytic and endocytic pathways. The process simultaneously moves thousands of different proteins efficiently and precisely between different compartments. And as if that weren�t enough - Intracellular transport must be able to respond to environmental and organismal conditions!!! Taken from the textbook.Taken from the textbook.