Cell Fractionation and the Cellular Response to Infection
Some pathogenic bacteria enter cells, replicate, and spread to other cells. Host cells respond by activating enzymatic pathways (Figure 1). Cells produce a steady supply of inactive caspase-1. In response to intracellular pathogens, inactive caspase-1 is cleaved to active caspase-1 (step 1). Active caspase-1 cleaves two other proteins: an inactive interleukin becomes an active signaling interleukin that is released from the cell (step 2), and cleaved gasdermin N-terminal fragments associate in the cell membrane to form large, nonspecific pores (step 3). Researchers used cell fractionation to determine where proteins are located. Proteins of known location were used as markers. The table shows which proteins are present in specific fractions.
| Aconitase (Krebs cycle) | DNA polymerase | GAPDH (glycolytic) | Sodium-potassium pump | NF-κB (immune response) | |
|---|---|---|---|---|---|
| Whole cell sample | + | + | + | + | + |
| Fraction 1 | + | ||||
| Fraction 2 | + | + | |||
| Fraction 3 | + | + | |||
| Fraction 4 | + |
(a)Using the marker proteins, identify the cell structure most likely enriched in Fraction 1 and in Fraction 4, and justify each identification. [2 pts]
(b)NF-κB appears in both Fraction 2 and Fraction 3. Explain how a single protein can be detected in two different fractions. [2 pts]
(c)Using Figure 1, explain how gasdermin pore formation would affect the ability of the infected cell to maintain homeostasis. [2 pts]
(d)Predict the effect on the immune response if a mutation prevented cleavage of the inactive interleukin, and justify your prediction. [2 pts]