Interfacial activation-based molecular (bio)-imprinting (IAMI) has been developed to rationally improve the performance of lipolytic enzymes in nonaqueous environments. The strategy combinedly exploits (i) the known dramatic enhancement of the protein conformational rigidity in a water-restricted milieu and (ii) the reported conformational changes associated with the activation of these enzymes at lipid-water interfaces, which basically involves an increased substrate accessibility to the active site and/or an induction of a more competent catalytic machinery. Six model enzymes have been assayed in several model reactions in nonaqueous media. The results, rationalized in light of the present biochemical and structural knowledge, show that the IAMI approach represents a straightforward, versatile method to generate manageable, activated (kinetically trapped) forms of lipolytic enzymes, providing under optimal conditions nonaqueous rate enhancements of up to two orders of magnitude. It is also shown that imprintability of lipolytic enzymes depends not only on the nature of the enzyme but also on the "quality" of the interface used as the template.

Plant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall, forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking. Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network, NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected. We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly, TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant, and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical, cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV. Plant viruses may use different host cell transport machineries to move from one cell to another through plasmodesmata. The contribution of host cell transport systems to the intercellular movement of multipartite negative-strand RNA plant viruses including tospoviruses is poorly understood. We used Tomato spotted wilt tospovirus (TSWV) as a model to understand the mechanism of intercellular movement of tospoviruses. In this study, using in vitro and in vivo systems for characterizing membrane proteins, we identified that the TSWV NSm movement protein was physically associated with the ER membrane. NSm expressed in a single leaf cell was able to move into neighboring cells along the ER membrane network. The ER membrane in plants is a unique structure that runs between neighboring cells via the ER desmotubule of the plasmodesmata and forms a continuous network throughout the plant. Taking advantage of TSWV NSm being tightly associated with ER membrane and trafficked between cells through plasmodesmata, we demonstrated here by robust biochemical, cellullar and genetic evidence that the ER membrane transport system of plants serves as an important route for intercellular trafficking of the NSm movement protein and TSWV. Our findings have important new implications for mechanistic studies on intercellular trafficking of tospoviruses and other multipartite negative-strand RNA plant viruses.

N‐glycosylation is the most common and versatile protein modification. In eukaryotic cells, this modification is catalyzed cotranslationally by the enzyme oligosaccharyltransferase, which targets the β‐amide of the asparagine in an Asn‐Xaa‐Ser/Thr consensus sequon (where Xaa is any amino acid but proline) in nascent proteins as they enter the endoplasmic reticulum. Because modification of the glycosylation acceptor site on membrane proteins occurs in a compartment‐specific manner, the presence of glycosylation is used to indicate membrane protein topology. Moreover, glycosylation sites can be added to gain topological information. In this study, we explored the determinants of N‐glycosylation with the in vitro transcription/translation of a truncated model protein in the presence of microsomes and surveyed 25,488 glycoproteins, of which 2,533 glycosylation sites had been experimentally validated. We found that glycosylation efficiency was dependent on both the distance to the C‐terminus and the nature of the amino acid that preceded the consensus sequon. These findings establish a broadly applicable method for membrane protein tagging in topological studies.

Proteins of the B‐cell lymphoma protein 2 (Bcl2) family are key regulators of the apoptotic cascade, controlling the release of apoptotic factors from the mitochondrial intermembrane space. A helical hairpin found in the core of water‐soluble folds of these proteins has been reported to be the pore‐forming domain. Here we show that peptides including any of the two α‐helix fragments of the hairpin of Bcl2 associated protein X (Bax) can independently induce release of large labelled dextrans from synthetic lipid vesicles. The permeability promoted by these peptides is influenced by intrinsic monolayer curvature and accompanied by fast transbilayer redistribution of lipids, supporting a toroidal pore mechanism as in the case of the full‐length protein. However, compared with the pores made by complete Bax, the pores made by the Bax peptides are smaller and do not need the concerted action of tBid. These data indicate that the sequences of both fragments of the hairpin contain the principal physicochemical requirements for pore formation, showing a parallel between the permeabilization mechanism of a complex regulated protein system, such as Bax, and the much simpler pore‐forming antibiotic peptides.