Konomi Fujimura-Kamada

Distortion of proximodistal information causes JNK-dependent apoptosis in Drosophila wing

Distinct and evolutionarily conserved signal-transduction cascades mediate the survival or death of cells during development. The c-Jun amino-terminal kinases (JNKs) of the mitogen-activated protein kinase superfamily are involved in apoptotic signalling in various cultured cells. However, the role of the JNK pathway in development is less well understood. In Drosophila, Decapentaplegic (Dpp; a homologue of transforming growth factor-β) and Wingless (Wg; a Wnt homologue) proteins are secretory morphogens that act cooperatively to induce formation of the proximodistal axis of appendages. Here we show that either decreased Dpp signalling in the distal wing cells or increased Dpp signalling in the proximal wing cells causes apoptosis. Inappropriate levels of Dpp signalling lead to aberrant morphogenesis in the respective wing zones, and these apoptotic zones are also determined by the strength of the Wg signal. Our results indicate that distortion of the positional information determined by Dpp and Wg signalling gradients leads to activation of the JNK apoptotic pathway, and the consequent induction of cell death thereby maintains normal morphogenesis.

A Drosophila MAPKKK, D‐MEKK1, mediates stress responses through activation of p38 MAPK

In cultured mammalian cells, the p38 mitogen‐activated protein kinase (MAPK) pathway is activated in response to a variety of environmental stresses. How ever, there is little evidence from in vivo studies to demonstrate a role for this pathway in the stress response. We identified a Drosophila MAPK kinase kinase (MAPKKK), D‐MEKK1, which can activate p38 MAPK. D‐MEKK1 is structurally similar to the mammalian MEKK4/MTK1 MAPKKK. D‐MEKK1 kinase activity was activated in animals under conditions of high osmolarity. Drosophila mutants lacking D‐MEKK1 were hypersensitive to environmental stresses, including elevated temperature and increased osmolarity. In these D‐MEKK1 mutants, activation of Drosophila p38 MAPK in response to stress was poor compared with activation in wild‐type animals. These results suggest that D‐MEKK1 regulation of the p38 MAPK pathway is critical for the response to environmental stresses in Drosophila.

Functions of phospholipid flippases

Asymmetrical distribution of phospholipids is generally observed in the eukaryotic plasma membrane. Maintenance and changes of this phospholipid asymmetry are regulated by ATP-driven phospholipid translocases. Accumulating evidence indicates that type 4 P-type ATPases (P4-ATPases, also called flippases) translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the plasma membrane and internal membranes. Among P-type ATPases, P4-ATPases are unique in that they are associated with a conserved membrane protein of the Cdc50 family as a non-catalytic subunit. Recent studies indicate that flippases are involved in various cellular functions, including transport vesicle formation and cell polarity. In this review, we will focus on the functional aspect of phospholipid flippases.

The upstream regulator, Rsr1p, and downstream effectors, Gic1p and Gic2p, of the Cdc42p small GTPase coordinately regulate initiation of budding in Saccharomyces cerevisiae

Background: Cdc42p, a Rho family small GTPase, is essential for budding initiation in the yeast Saccharomyces cerevisiae. The homologous proteins Gic1p and Gic2p (Gic1/2p) are effectors of Cdc42p, but their precise functions remain unknown. Rsr1p/Bud1p is a Ras family small GTPase that controls the selection of the budding site. Previous observations suggested that Rsr1p‐GTP recruits Cdc24p, a GDP/GTP exchange factor for Cdc42p, at the incipient bud site. However, this model only addresses how Rsr1p determines the budding site, because the rsr1 mutant normally initiates budding.

Isolation and characterization of novel mutations in CDC50, the non-catalytic subunit of the Drs2p phospholipid flippase.

Flippases (type 4 P-type ATPases) are believed to translocate phospholipids from the exoplasmic to the cytoplasmic leaflet in bilayer membranes. Since flippases are structurally similar to ion-transporting P-type ATPases such as the Ca(2+) ATPase, one important question is how flippases have evolved to transport phospholipids instead of ions. We previously showed that a conserved membrane protein, Cdc50p, is required for the endoplasmic reticulum exit of the Drs2p flippase in yeast. However, Cdc50p is still associated with Drs2p after its transport to the endosomal/trans-Golgi network (TGN) membranes, and its function in the complex with Drs2p is unknown. In this study, we isolated novel temperature-sensitive (ts) cdc50 mutants whose products were still localized to endosomal/TGN compartments at the non-permissive temperature. Mutant Cdc50 proteins colocalized with Drs2p in endosomal/TGN compartments, and they co-immunoprecipitated with Drs2p. These cdc50-ts mutants exhibited defects in vesicle transport from early endosomes to the TGN as the cdc50 deletion mutant did. These results suggest that mutant Cdc50 proteins could be complexed with Drs2p, but the resulting Cdc50p-Drs2p complex is functionally defective at the non-permissive temperature. Cdc50p may play an important role for phospholipid translocation by Drs2p.

Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid flippases in Saccharomyces cerevisiae

In eukaryotic cells, phosphatidylserine (PS) is predominantly located in the cytosolic leaflet of the plasma membrane; this asymmetry is generated by an unknown mechanism. In this study, we used the PS‐specific probe mRFP‐Lact‐C2 to investigate the possible involvement of type 4 P‐type ATPases, also called phospholipid flippases, in the generation of this asymmetry in Saccharomyces cerevisiae. PS was not found in the trans‐Golgi Network in wild‐type cells, but it became exposed when vesicle formation was compromised in the sec7 mutant, and it was also exposed on secretory vesicles (SVs), as reported previously. However, flippase mutations did not reduce the exposure of PS in either case, even at low levels that would only be detectable by quantitative analysis of mRFP‐Lact‐C2 fluorescence in isolated SVs. Furthermore, no reduction in the PS level was observed in a mutant with multiple flippase mutations. Because PS was not exposed in a mutant that accumulates ER or cis/medial‐Golgi membranes, Golgi maturation seems to be a prerequisite for PS translocation. Our results suggest that an unknown mechanism, possibly a protein with flippase‐like activity, acts in conjunction with known flippases to regulate PS translocation.

Interaction of the phospholipid flippase Drs2p with the F-box protein Rcy1p plays an important role in early endosome to trans-Golgi network vesicle transport in yeast

Phospholipid composition of biological membranes differs between the cytoplasmic and exoplasmic leaflets. The type 4 P-type ATPases are phospholipid flippases that generate such membrane phospholipid asymmetry. Drs2p, a flippase in budding yeast, is involved in the endocytic recycling pathway. Drs2p is implicated in clathrin-coated vesicle formation, but the underlying mechanisms are not clearly understood. Here we show that the carboxyl-terminal cytoplasmic region of Drs2p directly binds to Rcy1p, an F-box protein that is also required for endocytic recycling. The Drs2p-binding region was mapped to the amino acids 574-778 region of Rcy1p and a mutant Rcy1p lacking this region was defective in endocytic recycling of a v-SNARE Snc1p. We isolated Drs2p point mutants that reduced the interaction with Rcy1p. The mutation sites were clustered within a small region (a.a. 1260-1268) of Drs2p. Although these point mutants did not exhibit clear phenotypes, combination of them resulted in cold-sensitive growth, defects in endocytic recycling of Snc1p and defective localization of Rcy1p to endosomal membranes like the drs2 null mutant. These results suggest that the interaction of Drs2p with Rcy1p plays an important role for Drs2p function in the endocytic recycling pathway.

Essential Role of the NH2-Terminal Region of Cdc24 Guanine Nucleotide Exchange Factor in Its Initial Polarized Localization in Saccharomyces cerevisiae

ABSTRACT The cortical recruitment and accumulation of the small GTPase Cdc42 are crucial steps in the establishment of polarity, but this process remains obscure. Cdc24 is an upstream regulator of budding yeast Cdc42 that accelerates the exchange of GDP for GTP in Cdc42 via its Dbl homology (DH) domain. Here, we isolated five novel temperature-sensitive (ts) cdc24 mutants, the green fluorescent protein (GFP)-fused proteins of which lose their polarized localization at the nonpermissive temperature. All amino acid substitutions in the mutants were mapped to the NH2-terminal region of Cdc24, including the calponin homology (CH) domain. These Cdc24-ts mutant proteins did not interact with Bem1 at the COOH-terminal PB1 domain, suggesting a lack of exposure of the PB1 domain in the mutant proteins. The cdc24-ts mutants were also defective in polarization in the absence of Bem1. It was previously reported that a fusion protein containing Cdc24 and the p21-activated kinase (PAK)-like kinase Cla4 could bypass the requirement for Bem1 in polarity cue-independent budding (i.e., symmetry breaking). Cdc24-ts–Cla4 fusion proteins also showed ts localization at the polarity site. We propose that the NH2-terminal region unmasks the DH and PB1 domains, leading to the activation of Cdc42 and interaction with Bem1, respectively, to initiate cell polarization.

Cfs1p, a Novel Membrane Protein in the PQ-Loop Family, Is Involved in Phospholipid Flippase Functions in Yeast

Type 4 P-type ATPases (P4-ATPases) function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer, to generate and maintain asymmetric distribution of phospholipids at the plasma membrane and endosomal/Golgi membranes. The budding yeast Saccharomyces cerevisiae has four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), associated with the Cdc50p family noncatalytic subunit, and one monomeric flippase, Neo1p. They have been suggested to function in vesicle formation in membrane trafficking pathways, but details of their mechanisms remain to be clarified. Here, to search for novel factors that functionally interact with flippases, we screened transposon insertional mutants for strains that suppressed the cold-sensitive growth defect in the cdc50Δ mutant. We identified a mutation of YMR010W encoding a novel conserved membrane protein that belongs to the PQ-loop family including the cystine transporter cystinosin and the SWEET sugar transporters. We named this gene CFS1 (cdc fifty suppressor 1). GFP-tagged Cfs1p was partially colocalized with Drs2p and Neo1p to endosomal/late Golgi membranes. Interestingly, the cfs1Δ mutation suppressed growth defects in all flippase mutants. Accordingly, defects in membrane trafficking in the flippase mutants were also suppressed. These results suggest that Cfs1p and flippases function antagonistically in membrane trafficking pathways. A growth assay to assess sensitivity to duramycin, a phosphatidylethanolamine (PE)-binding peptide, suggested that the cfs1Δ mutation changed PE asymmetry in the plasma membrane. Cfs1p may thus be a novel regulator of phospholipid asymmetry.