Many virulence factors secreted from pathogenic Gram-negative bacteria are autotransporter proteins. The final step of autotransporter secretion is C 3 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 ␤-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 ␤-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 ␤-helix might serve as a template for the formation of native protein during OM secretion; hence, vectorial folding of the ␤-helix could contribute to the energyindependent translocation mechanism. Coupled with the sequence analysis, the results presented here suggest a general mechanism for autotransporter secretion.parallel ␤-sheet ͉ contact order ͉ outer membrane protein ͉ protein structure prediction ͉ virulence factor

Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption.

In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology.

We introduced pairs of cysteine residues into the passenger sequence of pertactin, an AT virulence protein from Bordetella pertussis, and show that OM secretion of the passenger domain stalls due to the formation of a disulphide bond. We further show that the C-terminus of the pertactin passenger domain b-helix crosses the OM first, followed by the N-terminal portions of the virulence protein. In vivo proteolytic digestion shows that the C-terminus is exposed to the extracellular milieu during stalling, and forms stable structure. These AT secretion and folding features can potentially facilitate efficient secretion.