Analysis of exocytosis mutants indicates close coupling between regulated secretion and transcription activation in Tetrahymena

Stimulation of regulated secretory cells promotes protein release via the fusion of cytoplasmic storage vesicles with the plasma membrane. In Tetrahymena thermophila, brief exposure to secretagogue results in synchronous fusion of the entire set of docked dense-core granules with the plasma membrane. We show that stimulation is followed by rapid new dense-core granule synthesis involving gene induction. Two genes encoding granule matrix proteins, GRL1 and GRL4, are shown to undergo induction following stimulation, resulting in ≈10-fold message accumulation within 1 h. The mechanism of induction involves transcriptional regulation, and the upstream region of GRL1 functions in vivo as an inducible promoter in a heterologous reporter construct using the gene encoding green fluorescent protein. Taking advantage of the characterized exocytosis (exo−) mutants available in this system, we asked whether the signals for regranulation were generated directly by the initial stimulation, or whether downstream events were required for transcription activation. Three mutants, with defects at three distinct stages in the regulated secretory pathway, failed to show induction of GRL1 and GRL4 after exposure to secretagogue. These results argue that regranulation depends upon signals generated by the final steps in exocytosis.

Differential use of the signal recognition particle translocase targeting pathway for inner membrane protein assembly in Escherichia coli

Assembly of several inner membrane proteins—leader peptidase (Lep), a Lep derivative (Lep-inv) that inserts with an inverted topology compared with the wild-type protein, the phage M13 procoat protein, and a procoat derivative (H1-procoat) with the hydrophobic core of the signal peptide replaced by a stretch from the first transmembrane segment in Lep—has been studied in vitro and in Escherichia coli strains that are conditional for the expression of either the 54 homologue (Ffh) or 4.5S RNA, which are the two components of the E. coli signal recognition particle (SRP), or SecE, an essential core component of the E. coli preprotein translocase. Membrane insertion has also been tested in a SecB null strain. Lep, Lep-inv, and H1-procoat require SRP for correct assembly into the inner membrane; in contrast, we find that wild-type procoat does not. Lep and, surprisingly, Lep-inv and H1-procoat fail to insert properly when SecE is depleted, whereas insertion of wild-type procoat is unaffected under these conditions. None of the proteins depend on SecB for assembly. These observations indicate that inner membrane proteins can assemble either by a mechanism in which SRP delivers the protein at the preprotein translocase or by what appears to be a direct integration into the lipid bilayer. The observed change in assembly mechanism when the hydrophobicity of the procoat signal peptide is increased demonstrates that the assembly of an inner membrane protein can be rerouted between different pathways.

Gap junctional coupling in lenses lacking α3 connexin

Fiber cells of the lens are interconnected by an extensive network of gap junctions containing α3 (Cx46) and α8 (Cx50) connexins. A specific role for these connexins in lens homeostasis is not known. To determine the contribution of these connexins to lens function, we used impedance techniques to study cell-to-cell coupling in lenses from homozygous α3 knockout (−/−), heterozygous (+/−), and wild-type (+/+) mice. Western blots and immunofluorescence data indicated that α8 remained at similar levels in the three classes of lenses, whereas α3 was approximately 50% of the normal level in the +/− lenses, and it was absent from the −/− lenses. Moreover, the data from +/+ lenses suggest that a cleavage of connexins occurs abruptly between the peripheral shell of differentiating fibers (DF) and the inner core of mature fibers (MF). The appearance of the cleaved connexins was correlated to a change in the coupling conductance. In −/− lenses the coupling conductance of MF was zero, and these fibers were depolarized by about 30 mV from normal (≈−65 mV). The DF remained coupled, but the conductance was reduced to 30–35% of normal. However, the gap junctions in the DF of α3 −/− lenses remained sensitive to pH. We conclude that α3 connexin is necessary for the coupling of central fibers to peripheral cells, and that this coupling is essential for fiber cell homeostasis because uncoupled MF depolarize and subsequently become opaque.