Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc, and actin gene transcription.

Stimulation of quiescent 3T3 cells with purified growth factors or of the pheochromocytoma cell line PC12 with nerve growth factor results in the rapid transient induction of c-fos, c-myc, and actin gene transcription (M.E. Greenberg and E.B. Ziff, Nature [London] 312:711-716; M.E. Greenberg, L.A. Greene, and E.B. Ziff, J. Biol. Chem. 26:14101-14110). We used protein synthesis inhibitors to investigate whether synthesis of new proteins plays a role in the rapid induction and subsequent repression of the transcription of these genes. Pretreatment of quiescent 3T3 cells with the inhibitor anisomycin before growth factor stimulation caused a superinduction of c-fos and c-myc mRNA levels upon growth factor addition. Nuclear runoff transcription analyses of 3T3 cells indicated that anisomycin potentiated c-fos, c-myc, and also actin expression at the transcriptional level, possibly by inhibiting transcriptional repression. Somewhat different results were obtained when PC12 cells were incubated with either anisomycin or cycloheximide. In PC12 cells protein synthesis inhibitors superinduced nerve growth factor activation of c-fos mRNA production but completely abolished the activation of c-myc. The results suggest that in PC12 cells c-fos transcription is activated by a protein-synthesis-independent mechanism, whereas c-myc stimulation requires new protein synthesis. The difference in the effect of anisomycin on growth factor activation of c-myc expression in 3T3 versus PC12 cells may be due to differential stringency of protein synthesis inhibition in the two cells or could reflect cell type differences in c-myc regulation.

Oligomerization of immunoglobulin G heavy and light chains in vitro. A cell-free assay to study the assembly of the endoplasmic reticulum.

A biochemical assay to study the assembly of the endoplasmic reticulum (ER) in a cell-free system is introduced. Incubation in vitro of ER vesicles containing only immunoglobulin gamma 1 heavy (H) chains with ER vesicles containing only K light (L) chains results in fusion and oligomerization of the H and L chains to form the H2L2 complex (immunoglobulin G). ER fusion/H2L2 oligomerization is time and temperature dependent and requires energy in the form of ATP. It is stimulated by the addition of cytosol and requires protease-sensitive components present on the membranes. The addition of guanosine 5'-O-(thiotriphosphate) inhibits membrane fusion and subsequent H2L2 oligomerization without affecting the assembly of H2L2 from detergent-solubilized pools, suggesting an important role for GTPases in vesicle recognition or fusion. The development of a rapid and quantitative assay to study the assembly of the ER in a cell-free system will allow us to identify components involved in the recognition, fusion, and post-fusion events critical for ER function in vivo.