Week 4
Major concept goals
-How a biochemical assay with purified mitochondria can be used to study the addition of proteins to this organelle
-How radioactivity labeling and SDS-PAGE can be used to follow specific proteins
-Protease protection can indicate that a protein is inside a membrane/organelle
-Distinguishing a result from a conclusion
-Creating and evaluating multiple conclusions from data
-Signal sequence hypothesis: that the amphipathic alpha helix at the N-term of proteins can cause the targeting and transport of proteins into mitochondria
-How charge, voltage, and Hsp ATPases function in the addition of proteins to mitochondria
-How Hsp chaperones act to transport proteins into mitochondria
-How a pulse-chase analysis of protein secretion informs how proteins are added to the Golgi
-How yeast mutants identified the machinery that mediates protein delivery to the Golgi
Overview of content
I. Adding proteins to mitochondria
A. The assay with non-energized mitochondria
1. The result
2. The conclusion
B The assay with energized mitochondria
1. The result
2. The conclusion
C. Data are facts and conclusions are what people think of the data (and are typically what are presented in a textbook)
D. The imported CoxIV is smaller, why?
E. CoxIV has an amphipathic alpha helix at its N-terminus, models for function?
F. Experiments to test the function of the amphipathic alpha helix at the N-terminus of CoxIV (2 types)
G. Correlation (association) experiments
H. Necessity (remove) experiments
I. Sufficiency (add) experiments
J. Mechanism of protein import to the matrix
a. TOM and TIM complexes
b. Unfolding in the cytoplasm
c. The function of the volatage gradient in moving the amphipathic alpha helix through the TIM complex into the matrix
d. The mechanisms by which the matrix Hsp70 mediates the import of the rest of the protein
K. Other pathways of protein targeting and transport in mitochondria
II. Making the Golgi
A. Golgi structure and function
B. Using a pulse-chase experiment to analyze how proteins are added to the Golgi
C. Using yeast mutants to analyze how proteins are added to the Golgi