Pseudohyphal growth in Saccharomyces cerevisiae involves protein kinase-regulated lipid flippases.

Lipid flippases of the P4 ATPase family establish phospholipid asymmetry in eukaryotic cell membranes and are involved in many essential cellular processes. The yeast Saccharomyces cerevisiae contains five P4 ATPases, among which Dnf3p is poorly characterized. Here, we demonstrate that Dnf3p is a flippase that catalyzes translocation of major glycerophospholipids, including phosphatidylserine, towards the cytosolic membrane leaflet. Deletion of the genes encoding Dnf3p and the distantly related P4 ATPases Dnf1p and Dnf2p results in yeast mutants with aberrant formation of pseudohyphae, suggesting that the Dnf1p-Dnf3p proteins have partly redundant functions in the control of this specialized form of polarized growth. Furthermore, as previously demonstrated for Dnf1 and Dnf2p, the phospholipid flipping activity of Dnf3p is positively regulated by flippase kinase 1 (Fpk1p) and Fpk2p. Phylogenetic analyses demonstrate that Dnf3p belongs to a subfamily of P4 ATPases specific for fungi and are likely to represent a hallmark of fungal evolution.

A component of the Sec61 ER protein transporting pore is required for plant susceptibility to powdery mildew.

Biotrophic pathogens, like the powdery mildew fungi, require living plant cells for their growth and reproduction. During infection, a specialized structure called the haustorium is formed by the fungus. The haustorium is surrounded by a plant cell-derived extrahaustorial membrane (EHM). Over the EHM, the fungus obtains nutrients from and secretes effector proteins into the plant cell. In the plant cell these effectors interfere with cellular processes such as pathogen defense and membrane trafficking. However, the mechanisms behind effector delivery are largely unknown. This paper provides a model for and new insights into a putative transfer mechanism of effectors into the plant cell. We show that silencing of the barley Sec61ßa transcript results in decreased susceptibility to the powdery mildew fungus. HvSec61ßa is a component of both the endoplasmic reticulum (ER) translocon and retrotranslocon pores, the latter being part of the ER-associated protein degradation machinery. We provide support for a model suggesting that the retrotranslocon function of HvSec61ßa is required for successful powdery mildew fungal infection. HvSec61ßa-GFP and a luminal ER marker were co-localized to the ER, which was found to be in close proximity to the EHM around the haustorial body, but not the haustorial fingers. This differential EHM proximity suggests that the ER, including HvSec61ßa, may be actively recruited by the haustorium, potentially to provide efficient effector transfer to the cytosol. Effector transport across this EHM-ER interface may occur by a vesicle-mediated process, while the Sec61 retrotranslocon pore potentially provides an escape route for these proteins to reach the cytosol.

Bimolecular fluorescence complementation and interaction of various Arabidopsis major intrinsic proteins expressed in yeast.

Tonoplast intrinsic proteins (TIPs) and plasma membrane intrinsic proteins (PIPs) form subgroups of plant major intrinsic proteins (MIPs) that channel water as well as various small neutral molecules across the tonoplast and plasma membrane. Most MIPs are believed to form homotetramers, while some plant PIPs have been shown to form heterotetramers composed of different isoforms. This study investigated in vivo molecular interactions between different Arabidopsis TIP isoforms and between TIPs and a PIP member. The interactions were assayed by bimolecular fluorescence complementation optimized for use in Saccharomyces cerevisiae as a heterologous expression system. Fluorescence of re-assembled Venus yellow fluorescent protein was monitored by fluorescence microscopy and flow cytometry. The results showed strong interactions between TIP1;2, TIP2;1 and TIP3;1. Surprisingly, the three TIP isoforms also interacted with PIP2;1. The potassium channel AKT1 was used as a negative control and exhibited no interaction with any of the MIPs. The observed interactions may play a role in targeting and regulation of MIPs in plants.

Interaction of barley powdery mildew effector candidate CSEP0055 with the defence protein PR17c.

A large number of effector candidates have been identified recently in powdery mildew fungi. However, their roles and how they perform their functions remain unresolved. In this study, we made use of host-induced gene silencing and confirmed that the secreted barley powdery mildew effector candidate, CSEP0055, contributes to the aggressiveness of the fungus. This result suggests that CSEP0055 is involved in the suppression of plant defence. A yeast two-hybrid screen indicated that CSEP0055 interacts with members of the barley pathogenesis-related protein families, PR1 and PR17. Interaction with PR17c was confirmed by bimolecular fluorescence complementation analyses. Down-regulation and over-expression of PR17c in epidermal cells of barley confirmed that this protein is important for penetration resistance against the powdery mildew fungus. In line with this, PR17c was found to be apoplastic, localizing to the papillae formed in response to this fungus. The CSEP0055 transcript did not start to accumulate until 24 h after inoculation. This suggests that this gene is expressed too late to influence primary penetration events, but rather sustains the fungus at sites of secondary penetration, where PR17c appears to be able to accumulate.

Intracellular targeting signals and lipid specificity determinants of the ALA/ALIS P4-ATPase complex reside in the catalytic ALA alpha-subunit.

Members of the P(4) subfamily of P-type ATPases are believed to catalyze flipping of phospholipids across cellular membranes, in this way contributing to vesicle biogenesis in the secretory and endocytic pathways. P(4)-ATPases form heteromeric complexes with Cdc50-like proteins, and it has been suggested that these act as beta-subunits in the P(4)-ATPase transport machinery. In this work, we investigated the role of Cdc50-like beta-subunits of P(4)-ATPases for targeting and function of P(4)-ATPase catalytic alpha-subunits. We show that the Arabidopsis P(4)-ATPases ALA2 and ALA3 gain functionality when coexpressed with any of three different ALIS Cdc50-like beta-subunits. However, the final cellular destination of P(4)-ATPases as well as their lipid substrate specificity are independent of the nature of the ALIS beta-subunit they were allowed to interact with.

Identification of a novel mouse P4-ATPase family member highly expressed during spermatogenesis.

P4-ATPases are transmembrane proteins unique to eukaryotes that play a fundamental role in vesicular transport. They have been proposed to act as phospholipid flippases thereby regulating lipid topology in cellular membranes. We cloned and characterized a novel murine P4-ATPase that is specifically expressed in testis, and named it FetA (flippase expressed in testis splicing form A). When expressed in Saccharomyces cerevisiae, FetA localizes partially to the plasma membrane resulting in increased internalization of NBD-labeled phosphatidylethanolamine and phosphatidylcholine, supporting a role for FetA in the inward lipid translocation across cellular membranes. In mouse testis, FetA protein is detected in gamete cells, from pachytene spermatocytes to mature sperms, and its intracellular localization is tightly related with acrosome formation, a process that involves intensive intracellular vesicle formation and fusion. Furthermore, loss-of-function of FetA by RNA interference in mastocytoma P815 cells profoundly perturbs the structural organization of the Golgi complex and causes loss of constitutive secretion at lower temperature. Our findings point to an essential role of FetA in Golgi morphology and secretory function, suggesting a crucial role for this novel murine P4-ATPase in spermatogenesis.