UV-A/B radiation rapidly activates photoprotective mechanisms in Chlamydomonas reinhardtii.

Conversion of light energy into chemical energy through photosynthesis in the chloroplasts of photosynthetic organisms is essential for photoautotrophic growth, and non-photochemical quenching (NPQ) of excess light energy prevents the generation of reactive oxygen species and maintains efficient photosynthesis under high light. In the unicellular green alga Chlamydomonas reinhardtii, NPQ is activated as a photoprotective mechanism through wavelength-specific light signaling pathways mediated by the phototropin (blue light) and ultra-violet (UV) light photoreceptors, but the biological significance of photoprotection activation by light with different qualities remains poorly understood. Here, we demonstrate that NPQ-dependent photoprotection is activated more rapidly by UV than by visible light. We found that induction of gene expression and protein accumulation related to photoprotection was significantly faster and greater in magnitude under UV treatment compared with that under blue- or red-light treatment. Furthermore, the action spectrum of UV-dependent induction of photoprotective factors implied that C. reinhardtii senses relatively long-wavelength UV (including UV-A/B), whereas the model dicot plant Arabidopsis (Arabidopsis thaliana) preferentially senses relatively short-wavelength UV (mainly UV-B/C) for induction of photoprotective responses. Therefore, we hypothesize that C. reinhardtii developed a UV response distinct from that of land plants.

The CONSTANS flowering complex controls the protective response of photosynthesis in the green alga Chlamydomonas.

Light is essential for photosynthesis, but the amounts of light that exceed an organism's assimilation capacity can result in oxidative stress and even cell death. Plants and microalgae have developed a photoprotective response mechanism, qE, that dissipates excess light energy as thermal energy. In the green alga Chlamydomonas reinhardtii, qE is regulated by light-inducible photoprotective proteins, but the pathway from light perception to qE is not fully understood. Here, we show that the transcription factors CONSTANS and Nuclear transcription Factor Ys (NF-Ys) form a complex that governs light-dependent photoprotective responses in C. reinhardtii. The qE responses do not occur in CONSTANS or NF-Y mutants. The signal from light perception to the CONSTANS/NF-Ys complex is directly inhibited by the SPA1/COP1-dependent E3 ubiquitin ligase. This negative regulation mediated by the E3 ubiquitin ligase and the CONSTANS/NF-Ys complex is common to photoprotective response in algal photosynthesis and flowering in plants.

Isolation of photoprotective signal transduction mutants by systematic bioluminescence screening in Chlamydomonas reinhardtii.

In photosynthetic organisms, photoprotection to avoid overexcitation of photosystems is a prerequisite for survival. Green algae have evolved light-inducible photoprotective mechanisms mediated by genes such as light-harvesting complex stress-related (LHCSR). Studies on the light-dependent regulation of LHCSR expression in the green alga Chlamydomonas reinhardtii have revealed that photoreceptors for blue light (phototropin) and ultraviolet light perception (UVR8) play key roles in initiating photoprotective signal transduction. Although initial light perception via phototropin or UVR8 is known to result in increased LHCSR3 and LHCSR1 gene expression, respectively, the mechanisms of signal transduction from the input (light perception) to the output (gene expression) remain unclear. In this study, to further elucidate the signal transduction pathway of the photoprotective response of green algae, we established a systematic screening protocol for UV-inducible LHCSR1 gene expression mutants using a bioluminescence reporter assay. Following random mutagenesis screening, we succeeded in isolating mutants deficient in LHCSR1 gene and protein expression after UV illumination. Further characterization revealed that the obtained mutants could be separated into 3 different phenotype groups, the "UV-specific", "LHCSR1-promoter/transcript-specific" and "general photoprotective" mutant groups, which provided further insight into photoprotective signal transduction in C. reinhardtii.

Algal photoprotection is regulated by the E3 ligase CUL4-DDB1DET1.

Light is essential for photosynthesis, but the amounts of light that exceed an organism's assimilation capacity can cause serious damage1. Photosynthetic organisms minimize such potential harm through protection mechanisms collectively referred to as non-photochemical quenching2. One mechanism of non-photochemical quenching called energy-dependent quenching (qE quenching) is readily activated under high-light conditions and dissipates excess energy as heat. LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEINS 1 and 3 (LHCSR1 and LHCSR3) have been proposed to mediate qE quenching in the green alga Chlamydomonas reinhardtii when grown under high-light conditions3. LHCSR3 induction requires a blue-light photoreceptor, PHOTOTROPIN (PHOT)4, although the signal transduction pathway between PHOT and LHCSR3 is not yet clear. Here, we identify two phot suppressor loci involved in qE quenching: de-etiolated 1 (det1)5 and damaged DNA-binding 1 (ddb1)6. Using a yeast two-hybrid analysis and an inhibitor assay, we determined that these two genetic elements are part of a protein complex containing CULLIN 4 (CUL4). These findings suggest a photoprotective role for the putative E3 ubiquitin ligase CUL4-DDB1DET1 in unicellular photosynthetic organisms that may mediate blue-light signals to LHCSR1 and LHCSR3 gene expression.

Phospholipid flippases and Sfk1p, a novel regulator of phospholipid asymmetry, contribute to low permeability of the plasma membrane.

Phospholipid flippase (type 4 P-type ATPase) plays a major role in the generation of phospholipid asymmetry in eukaryotic cell membranes. Loss of Lem3p-Dnf1/2p flippases leads to the exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell surface in yeast, resulting in sensitivity to PS- or PE-binding peptides. We isolated Sfk1p, a conserved membrane protein in the TMEM150/FRAG1/DRAM family, as a multicopy suppressor of this sensitivity. Overexpression of SFK1 decreased PS/PE exposure in lem3Δ mutant cells. Consistent with this, lem3Δ sfk1Δ double mutant cells exposed more PS/PE than the lem3Δ mutant. Sfk1p was previously implicated in the regulation of the phosphatidylinositol-4 kinase Stt4p, but the effect of Sfk1p on PS/PE exposure in lem3Δ was independent of Stt4p. Surprisingly, Sfk1p did not facilitate phospholipid flipping but instead repressed it, even under ATP-depleted conditions. We propose that Sfk1p negatively regulates transbilayer movement of phospholipids irrespective of directions. In addition, we showed that the permeability of the plasma membrane was dramatically elevated in the lem3Δ sfk1Δ double mutant in comparison with the corresponding single mutants. Interestingly, total ergosterol was decreased in the lem3Δ sfk1Δ mutant. Our results suggest that phospholipid asymmetry is required for the maintenance of low plasma membrane permeability.

Isolation of uracil auxotroph mutants of coral symbiont alga for symbiosis studies.

Coral reef ecosystems rely on stable symbiotic relationship between the dinoflagellate Symbiodinium spp. and host cnidarian animals. The collapse of such symbiosis could cause coral 'bleaching' and subsequent host death. Despite huge interest on Symbiodinium, lack of mutant strains and readily available genetic tools have hampered molecular research. A major issue was the tolerance to marker antibiotics. Here, we isolated Symbiodinium mutants requiring uracil for growth, and hence, useful in transformation screening. We cultured Symbiodinium spp. cells in the presence of 5-fluoroorotic acid (5FOA), which inhibits the growth of cells expressing URA3 encoding orotidine-5'-monophosphate decarboxylase, and isolated cells that require uracil for growth. Sequence analyses and genetic complementation tests using yeast demonstrated that one of the mutant cell lines had a point mutation in URA3, resulting in a splicing error at an unusual exon-intron junction, and consequently, loss of enzyme activity. This mutant could maintain a symbiotic relationship with the model sea anemone Exaiptasia pallida only in sea water containing uracil. Results show that the URA3 mutant will be a useful tool for screening Symbiodinium transformants, both ex and in hospite, as survival in the absence of uracil is possible only upon successful introduction of URA3.

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.

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.

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.

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.

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.

Transbilayer phospholipid flipping regulates Cdc42p signaling during polarized cell growth via Rga GTPase-activating proteins.

An important problem in polarized morphogenesis is how polarized transport of membrane vesicles is spatiotemporally regulated. Here, we report that a local change in the transbilayer phospholipid distribution of the plasma membrane regulates the axis of polarized growth. Type 4 P-type ATPases Lem3p-Dnf1p and -Dnf2p are putative heteromeric phospholipid flippases in budding yeast that are localized to polarized sites on the plasma membrane. The lem3Delta mutant exhibits prolonged apical growth due to a defect in the switch to isotropic bud growth. In lem3Delta cells, the small GTPase Cdc42p remains polarized at the bud tip where phosphatidylethanolamine remains exposed on the outer leaflet. Intriguingly, phosphatidylethanolamine and phosphatidylserine stimulate GTPase-activating protein (GAP) activity of Rga1p and Rga2p toward Cdc42p, whereas PI(4,5)P(2) inhibits it. We propose that a redistribution of phospholipids to the inner leaflet of the plasma membrane triggers the dispersal of Cdc42p from the apical growth site, through activation of GAPs.

Endocytic recycling in yeast is regulated by putative phospholipid translocases and the Ypt31p/32p-Rcy1p pathway.

Phospholipid translocases (PLTs) have been implicated in the generation of phospholipid asymmetry in membrane bilayers. In budding yeast, putative PLTs are encoded by the DRS2 gene family of type 4 P-type ATPases. The homologous proteins Cdc50p, Lem3p, and Crf1p are potential noncatalytic subunits of Drs2p, Dnf1p and Dnf2p, and Dnf3p, respectively; these putative heteromeric PLTs share an essential function for cell growth. We constructed temperature-sensitive mutants of CDC50 in the lem3Delta crf1Delta background (cdc50-ts mutants). Screening for multicopy suppressors of cdc50-ts identified YPT31/32, two genes that encode Rab family small GTPases that are involved in both the exocytic and endocytic recycling pathways. The cdc50-ts mutants did not exhibit major defects in the exocytic pathways, but they did exhibit those in endocytic recycling; large membranous structures containing the vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor Snc1p intracellularly accumulated in these mutants. Genetic results suggested that the YPT31/32 effector RCY1 and CDC50 function in the same signaling pathway, and simultaneous overexpression of CDC50, DRS2, and GFP-SNC1 restored growth as well as the plasma membrane localization of GFP-Snc1p in the rcy1Delta mutant. In addition, Rcy1p coimmunoprecipitated with Cdc50p-Drs2p. We propose that the Ypt31p/32p-Rcy1p pathway regulates putative phospholipid translocases to promote formation of vesicles destined for the trans-Golgi network from early endosomes.

Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast.

Lem3p-Dnf1p is a putative aminophospholipid translocase (APLT) complex that is localized to the plasma membrane; Lem3p is required for Dnf1p localization to the plasma membrane. We have identified lem3 mutations, which did not affect formation or localization of the Lem3p-Dnf1p complex, but caused a synthetic growth defect with the null mutation of CDC50, a structurally and functionally redundant homologue of LEM3. Interestingly, these lem3 mutants exhibited nearly normal levels of NBD-labeled phospholipid internalization across the plasma membrane, suggesting that Lem3p may have other functions in addition to regulation of the putative APLT activity of Dnf1p at the plasma membrane. Similarly, deletion of the COOH-terminal cytoplasmic region of Dnf1p affected neither the localization nor the APLT activity of Dnf1p at the plasma membrane, but caused a growth defect in the cdc50Delta background. Our results suggest that the Lem3p-Dnf1p complex may play a role distinct from its plasma membrane APLT activity when it substitutes for the Cdc50p-Drs2p complex, its redundant partner in the endosomal/trans-Golgi network compartments.

Cdc50p, a protein required for polarized growth, associates with the Drs2p P-type ATPase implicated in phospholipid translocation in Saccharomyces cerevisiae.

Cdc50p, a transmembrane protein localized to the late endosome, is required for polarized cell growth in yeast. Genetic studies suggest that CDC50 performs a function similar to DRS2, which encodes a P-type ATPase of the aminophospholipid translocase (APT) subfamily. At low temperatures, drs2Delta mutant cells exhibited depolarization of cortical actin patches and mislocalization of polarity regulators, such as Bni1p and Gic1p, in a manner similar to the cdc50Delta mutant. Both Cdc50p and Drs2p were localized to the trans-Golgi network and late endosome. Cdc50p was coimmunoprecipitated with Drs2p from membrane protein extracts. In cdc50Delta mutant cells, Drs2p resided on the endoplasmic reticulum (ER), whereas Cdc50p was found on the ER membrane in drs2Delta cells, suggesting that the association on the ER membrane is required for transport of the Cdc50p-Drs2p complex to the trans-Golgi network. Lem3/Ros3p, a homolog of Cdc50p, was coimmunoprecipitated with another APT, Dnf1p; Lem3p was required for exit of Dnf1p out of the ER. Both Cdc50p-Drs2p and Lem3p-Dnf1p were confined to the plasma membrane upon blockade of endocytosis, suggesting that these proteins cycle between the exocytic and endocytic pathways, likely performing redundant functions. Thus, phospholipid asymmetry plays an important role in the establishment of cell polarity; the Cdc50p/Lem3p family likely constitute potential subunits specific to unique P-type ATPases of the APT subfamily.

She4p/Dim1p interacts with the motor domain of unconventional myosins in the budding yeast, Saccharomyces cerevisiae.

She4p/Dim1p, a member of the UNC-45/CRO1/She4p (UCS) domain-containing protein family, is required for endocytosis, polarization of actin cytoskeleton, and polarization of ASH1 mRNA in Saccharomyces cerevisiae. We show herein that She4p/Dim1p is involved in endocytosis and actin polarization through interactions with the type I myosins Myo3p and Myo5p. Two-hybrid and biochemical experiments showed that She4p/Dim1p interacts with the motor domain of Myo3/5p through its UCS domain. She4p/Dim1p was required for Myo5p localization to cortical patch-like structures. Using random mutagenesis of the motor region of MYO5, we identified four independent dominant point mutations that suppress the temperature-sensitive growth phenotype of the she4/dim1 null mutant. All of the amino acid substitutions caused by these mutations, V164I, N168I, N209S, and K377M, could suppress the defects of endocytosis and actin polarization of the she4/dim1 mutant as well. She4p/Dim1p also showed two-hybrid interactions with the motor domain of a type II myosin Myo1p and type V myosins Myo2p and Myo4p, and was required for proper localization of Myo4p, which regulates polarization of ASH1 mRNA. Our results suggest that She4p/Dim1p is required for structural integrity or regulation of the motor domain of unconventional myosins.

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. RESULTS: Here we show that a rsr1 gic1 gic2 mutant fails to initiate budding, resulting in unbudded, large, and multinucleated cells. Expression of a dominant active or dominant negative mutant of RSR1 also inhibited the growth of the gic1 gic2 mutant, suggesting that cycling of Rsr1p between the GTP- and GDP-bound forms is required for budding initiation in the gic1 gic2 mutant. Among the mutations in effectors of CDC42, only the gic1 gic2 mutation demonstrated a synthetic lethal interaction with rsr1. Increased gene dosage of CDC42 suppressed defects in budding initiation of rsr1 gic1 gic2 mutants containing additional mutations in other effectors of CDC42, including BNI1, CLA4 or STE20. The polarized localization of Bni1p-GFP (green fluorescent protein) and Cla4p-GFP was lost after depletion of Gic1p in the rsr1 gic2 mutant. CONCLUSION: We propose that Gic1/2p may stabilize or maintain a complex consisting of Cdc42p-GTP and its effectors at the budding site, which are assembled by the action of the Rsr1p-Cdc24p system.

Cdc50p, a conserved endosomal membrane protein, controls polarized growth in Saccharomyces cerevisiae.

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.