14-3-3zeta escorts CCTalpha for calcium-activated nuclear import in lung epithelia.

Integrity of animal biomembranes is critical to preserve normal cellular functions and viability. Phosphatidylcholine, an indispensible membrane component, requires the enzyme CCTalpha for its biosynthesis. Nuclear expression of CCTalpha is needed for expansion of the nuclear membrane network, but mechanisms for CCTalpha nuclear import are unknown. Herein, we show that in epithelia, extracellular Ca(2+) triggers CCTalpha cytoplasmic-nuclear translocation. CCTalpha nuclear import was associated with binding to 14-3-3zeta, a key regulator of protein trafficking. 14-3-3zeta was both sufficient and required for CCTalpha nuclear import. Helix G within the 14-3-3zeta binding groove interacts with a putative molecular signature within the CCTalpha carboxyl-terminal phosphoserine motif (residues 328-343). 14-3-3zeta was critically involved in preserving phosphatidylcholine synthesis and cell viability in a model of Pseudomonas aeruginosa infection where Ca(2+) concentrations increase within epithelia. Thus, 14-3-3zeta controls CCTalpha nuclear import in response to calcium signals, thereby regulating mammalian phospholipid synthesis. Agassandian, M., Chen, B. B., Schuster, C. C., Houtman, J. C. D., Mallampalli, R. K. 14-3-3zeta escorts CCTalpha for calcium-activated nuclear import in lung epithelia.

Pertactin beta-helix folding mechanism suggests common themes for the secretion and folding of autotransporter proteins.

Many virulence factors secreted from pathogenic Gram-negative bacteria are autotransporter proteins. The final step of autotransporter secretion is C --> N-terminal threading of the passenger domain through the outer membrane (OM), mediated by a cotranslated C-terminal porin domain. The native structure is formed only after this final secretion step, which requires neither ATP nor a proton gradient. Sequence analysis reveals that, despite size, sequence, and functional diversity among autotransporter passenger domains, >97% are predicted to form parallel beta-helices, indicating this structural topology may be important for secretion. We report the folding behavior of pertactin, an autotransporter passenger domain from Bordetella pertussis. The pertactin beta-helix folds reversibly in isolation, but folding is much slower than expected based on size and native-state topology. Surprisingly, pertactin is not prone to aggregation during folding, even though folding is extremely slow. Interestingly, equilibrium denaturation results in the formation of a partially folded structure, a stable core comprising the C-terminal half of the protein. Examination of the pertactin crystal structure does not reveal any obvious reason for the enhanced stability of the C terminus. In vivo, slow folding would prevent premature folding of the passenger domain in the periplasm, before OM secretion. Moreover, the extra stability of the C-terminal rungs of the beta-helix might serve as a template for the formation of native protein during OM secretion; hence, vectorial folding of the beta-helix could contribute to the energy-independent translocation mechanism. Coupled with the sequence analysis, the results presented here suggest a general mechanism for autotransporter secretion.