Vesicles In Yeast Research Articles

Regulation of yeast polarized exocytosis by phosphoinositide lipids.

Phosphoinositides help steer membrane trafficking routes within eukaryotic cells. In polarized exocytosis, which targets vesicular cargo to sites of polarized growth at the plasma membrane (PM), the two phosphoinositides phosphatidylinositol 4-phosphate (PI4P) and its derivative phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) pave the pathway for vesicle transport from the Golgi to the PM. PI4P is a critical regulator of mechanisms that shape late Golgi membranes for vesicle biogenesis and release. Although enriched in vesicle membranes, PI4P is inexplicably removed from post-Golgi vesicles during their transit to the PM, which drives subsequent steps in exocytosis. At the PM, PI(4,5)P2 recruits effectors that establish polarized membrane sites for targeting the vesicular delivery of secretory cargo. The budding yeast Saccharomyces cerevisiae provides an elegant model to unravel the complexities of phosphoinositide regulation during polarized exocytosis. Here, we review how PI4P and PI(4,5)P2 promote yeast vesicle biogenesis, exocyst complex assembly and vesicle docking at polarized cortical sites, and suggest how these steps might impact related mechanisms of human disease.

Exploring Extracellular Vesicles of Probiotic Yeast as Carriers of Biologically Active Molecules Transferred to Human Intestinal Cells.

Extracellular vesicles (EVs) are nanoparticles containing various bioactive cargos-e.g., proteins, RNAs, and lipids-that are released into the environment by all cell types. They are involved in, amongst other functions, intercellular communication. This article presents studies on EVs produced by the probiotic yeast Saccharomyces boulardii CNCM I-745. The size distribution and concentration of EVs in the liquid culture of yeast were estimated. Moreover, the vesicles of S. boulardii were tested for their cytotoxicity against three model human intestinal cell lines. This study did not show any significant negative effect of yeast EVs on these cells under tested conditions. In addition, EVs of S. boulardii were verified for their ability to internalize in vitro with human cells and transfer their cargo. The yeast vesicles were loaded with doxorubicin, an anticancer agent, and added to the cellular cultures. Subsequently, microscopic observations revealed that these EVs transferred the compound to human intestinal cell lines. A cytotoxicity test confirmed the activity of the transferred doxorubicin. Detailed information about the proteins present in EVs might be important in terms of exploring yeast EVs as carriers of active molecules. Thus, proteomic analysis of the EV content was also conducted within the present study, and it allowed the identification of 541 proteins after matching them to the Saccharomyces Genome Database (SGD). Altogether, this study provides strong evidence that the EVs of the probiotic CNCM I-745 strain could be considered a drug delivery system.

Genome doubling enabled the expansion of yeast vesicle traffic pathways

Vesicle budding and fusion in eukaryotes depend on a suite of protein types, such as Arfs, Rabs, coats and SNAREs. Distinct paralogs of these proteins act at distinct intracellular locations, suggesting a link between gene duplication and the expansion of vesicle traffic pathways. Genome doubling, a common source of paralogous genes in fungi, provides an ideal setting in which to explore this link. Here we trace the fates of paralog doublets derived from the 100-Ma-old hybridization event that gave rise to the whole genome duplication clade of budding yeast. We find that paralog doublets involved in specific vesicle traffic functions and pathways are convergently retained across the entire clade. Vesicle coats and adaptors involved in secretory and early-endocytic pathways are retained as doublets, at rates several-fold higher than expected by chance. Proteins involved in later endocytic steps and intra-Golgi traffic, including the entire set of multi-subunit and coiled-coil tethers, have reverted to singletons. These patterns demonstrate that selection has acted to expand and diversify the yeast vesicle traffic apparatus, across species and time.

Identification of an Isoflavonoid Transporter Required for the Nodule Establishment of the Rhizobium-Fabaceae Symbiotic Interaction.

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by, for example, forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

Artificial ER-Derived Vesicles as Synthetic Organelles for in Vivo Compartmentalization of Biochemical Pathways.

Compartmentalization in membrane-surrounded organelles has the potential to overcome obstacles associated with the engineering of metabolic pathways, such as unwanted side reactions, accumulation of toxic intermediates, drain of intermediates out of the cell, and long diffusion distances. Strategies utilizing natural organelles suffer from the presence of endogenous pathways. In our approach, we make use of endoplasmic reticulum-derived vesicles loaded with enzymes of a metabolic pathway ("metabolic vesicles"). They are generated by fusion of synthetic peptides containing the N-terminal proline-rich and self-assembling region of the maize storage protein gamma-Zein ("Zera") to the pathway enzymes. We have applied a strategy to integrate three enzymes of a cis,cis-muconic acid production pathway into those vesicles in yeast. Using fluorescence microscopy and cell fractionation techniques, we have proven the formation of metabolic vesicles and the incorporation of enzymes. Activities of the enzymes and functionality of the compartmentalized pathway were demonstrated in fermentation experiments.

Yeast as a Model System to Study Trafficking of Small Vesicles Carrying Signal-less Proteins In and Out of the Cell.

Exosomes are small vesicles that are released from a variety of cells and are involved in cell-to-cell communication. In humans, exosomes are detected in the plasma, urine, saliva, and cerebrospinal fluid. These vesicles carry multiple cargo proteins, as well as microRNA that affect the transcription of target genes. During cancer progression, secretion of exosomes increases. As such, proteins and microRNAs released from exosomes in cancer patients can be used as biomarkers for cancer diagnosis and progression. Yeast is an excellent model system to study trafficking of small vesicles in and out of the cells. When yeast cells are grown in low glucose, small vesicles transport gluconeogenic enzymes to different locations. In the cytoplasm, they exist as free vesicles and aggregated clusters. They are also secreted and account for more than 90% of total organelles in the extracellular fraction. When glucose is added to prolongedstarved cells, extracellular vesicles are endocytosed resulting in a dramatic decrease in vesicles in the extracellular fraction and the appearance of vesicle clusters in the cytoplasm. Internalization also leads to a rapid decline in protein levels for vesicle-associated proteins in the extracellular fraction. Using large-scale proteomics/secretomics, more than 300 proteins that were secreted into the extracellular fraction/periplasm were identified. They showed distribution patterns similar to gluconeogenic enzymes. They were secreted during glucose starvation and their levels in the extracellular fraction decreased following glucose re-feeding. Multiple techniques have been used to study the biogenesis, clustering, secretion, and internalization of vesicles in yeast.

Arabidopsis COG Complex Subunits COG3 and COG8 Modulate Golgi Morphology, Vesicle Trafficking Homeostasis and Are Essential for Pollen Tube Growth.

Spatially and temporally regulated membrane trafficking events incorporate membrane and cell wall materials into the pollen tube apex and are believed to underlie the rapid pollen tube growth. In plants, the molecular mechanisms and physiological functions of intra-Golgi transport and Golgi integrity maintenance remain largely unclear. The conserved oligomeric Golgi (COG) complex has been implicated in tethering of retrograde intra-Golgi vesicles in yeast and mammalian cells. Using genetic and cytologic approaches, we demonstrate that T-DNA insertions in Arabidopsis COG complex subunits, COG3 and COG8, cause an absolute, male-specific transmission defect that can be complemented by expression of COG3 and COG8 from the LAT52 pollen promoter, respectively. No obvious abnormalities in the microgametogenesis of the two mutants are observed, but in vitro and in vivo pollen tube growth are defective. COG3 or COG8 proteins fused to green fluorescent protein (GFP) label the Golgi apparatus. In pollen of both mutants, Golgi bodies exhibit altered morphology. Moreover, γ-COP and EMP12 proteins lose their tight association with the Golgi. These defects lead to the incorrect deposition of cell wall components and proteins during pollen tube growth. COG3 and COG8 interact directly with each other, and a structural model of the Arabidopsis COG complex is proposed. We believe that the COG complex helps to modulate Golgi morphology and vesicle trafficking homeostasis during pollen tube tip growth.

Generic Sorting of Raft Lipids into Secretory Vesicles in Yeast

Previous work has showed that ergosterol and sphingolipids become sorted to secretory vesicles immunoisolated using a chimeric, artificial raft membrane protein as bait. In this study, we have extended this analysis to three populations of secretory vesicles isolated using natural yeast plasma membrane (PM) proteins: Pma1p, Mid2p and Gap1*p as baits. We compared the lipidomes of the immunoisolated vesicles with each other and with the lipidomes of the donor compartment, the trans-Golgi network, and the acceptor compartment, the PM, using a quantitative mass spectrometry approach that provided a complete lipid overview of the yeast late secretory pathway. We could show that vesicles captured with different baits carry the same cargo and have almost identical lipid compositions; being highly enriched in ergosterol and sphingolipids. This finding indicates that lipid raft sorting is a generic feature of vesicles carrying PM cargo and suggests a common lipid-based mechanism for their formation.

The AP-3 adaptor complex is required for vacuolar function in Arabidopsis

Subcellular trafficking is required for a multitude of functions in eukaryotic cells. It involves regulation of cargo sorting, vesicle formation, trafficking and fusion processes at multiple levels. Adaptor protein (AP) complexes are key regulators of cargo sorting into vesicles in yeast and mammals but their existence and function in plants have not been demonstrated. Here we report the identification of the protein-affected trafficking 4 (pat4) mutant defective in the putative δ subunit of the AP-3 complex. pat4 and pat2, a mutant isolated from the same GFP imaging-based forward genetic screen that lacks a functional putative AP-3 β, as well as dominant negative AP-3 μ transgenic lines display undistinguishable phenotypes characterized by largely normal morphology and development, but strong intracellular accumulation of membrane proteins in aberrant vacuolar structures. All mutants are defective in morphology and function of lytic and protein storage vacuoles (PSVs) but show normal sorting of reserve proteins to PSVs. Immunoprecipitation experiments and genetic studies revealed tight functional and physical associations of putative AP-3 β and AP-3 δ subunits. Furthermore, both proteins are closely linked with putative AP-3 μ and σ subunits and several components of the clathrin and dynamin machineries. Taken together, these results demonstrate that AP complexes, similar to those in other eukaryotes, exist in plants, and that AP-3 plays a specific role in the regulation of biogenesis and function of vacuoles in plant cells.

Biogenesis of extracellular vesicles in yeast

The cellular events required for unconventional protein secretion in eukaryotic pathogens are beginning to be revealed. In fungi, extracellular release of proteins involves passage through the cell wall by mechanisms that are poorly understood. In recent years, several studies demonstrated that yeast cells produce vesicles that traverse the cell wall to release a wide range of cellular components into the extracellular space. These studies suggested that extracellular vesicle release involves components of both conventional and unconventional secretory pathways, although the precise mechanisms required for this process are still unknown. We discuss here cellular events that are candidates for regulating this interesting but elusive event in the biology of yeast cells.

Grapevine MATE-type proteins act as vacuolar H+-dependent acylated anthocyanin transporters.

In grapevine (Vitis vinifera), anthocyanins are responsible for most of the red, blue, and purple pigmentation found in the skin of berries. In cells, anthocyanins are synthesized in the cytoplasm and accumulated into the vacuole. However, little is known about the transport of these compounds through the tonoplast. Recently, the sequencing of the grapevine genome allowed us to identify genes encoding proteins with high sequence similarity to the Multidrug And Toxic Extrusion (MATE) family. Among them, we selected two genes as anthocyanin transporter candidates and named them anthoMATE1 (AM1) and AM3. The expression of both genes was mainly fruit specific and concomitant with the accumulation of anthocyanin pigment. Subcellular localization assays in grapevine hairy roots stably transformed with AM1 or AM3green fluorescent protein fusion protein revealed that AM1 and AM3 are primarily localized to the tonoplast. Yeast vesicles expressing anthoMATEs transported acylated anthocyanins in the presence of MgATP. Inhibitor studies demonstrated that AM1 and AM3 proteins act in vitro as vacuolar H(+)-dependent acylated anthocyanin transporters. By contrast, under our experimental conditions, anthoMATEs could not transport malvidin 3-O-glucoside or cyanidin 3-O-glucoside, suggesting that the acyl conjugation was essential for the uptake. Taken together, these results provide evidence that in vitro the two grapevine AM1 and AM3 proteins mediate specifically acylated anthocyanin transport.

TheArabidopsisMATE Transporter TT12 Acts as a Vacuolar Flavonoid/H+-Antiporter Active in Proanthocyanidin-Accumulating Cells of the Seed Coat

Phenotypic characterization of the Arabidopsis thaliana transparent testa12 (tt12) mutant encoding a membrane protein of the multidrug and toxic efflux transporter family, suggested that TT12 is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed. Metabolite analysis in tt12 seeds reveals an absence of flavan-3-ols and proanthocyanidins together with a reduction of the major flavonol quercetin-3-O-rhamnoside. The TT12 promoter is active in cells synthesizing proanthocyanidins. Using translational fusions between TT12 and green fluorescent protein, it is demonstrated that this transporter localizes to the tonoplast. Yeast vesicles expressing TT12 can transport the anthocyanin cyanidin-3-O-glucoside in the presence of MgATP but not the aglycones cyanidin and epicatechin. Inhibitor studies demonstrate that TT12 acts in vitro as a cyanidin-3-O-glucoside/H(+)-antiporter. TT12 does not transport glycosylated flavonols and procyanidin dimers, and a direct transport activity for catechin-3-O-glucoside, a glucosylated flavan-3-ol, was not detectable. However, catechin-3-O-glucoside inhibited TT12-mediated transport of cyanidin-3-O-glucoside in a dose-dependent manner, while flavan-3-ol aglycones and glycosylated flavonols had no effect on anthocyanin transport. It is proposed that TT12 transports glycosylated flavan-3-ols in vivo. Mutant banyuls (ban) seeds accumulate anthocyanins instead of proanthocyanidins, yet the ban tt12 double mutant exhibits reduced anthocyanin accumulation, which supports the transport data suggesting that TT12 mediates anthocyanin transport in vitro.

A putative Sec23 homologue of Plasmodium falciparum is located in Maurer's clefts.

Erythrocytes infected with the human malarial para-site Plasmodium falciparum acquire adhesive propertiesand sequester in the microvasculature. This mechanism,referred to as cytoadhesion, involves the transport of par-asite encoded immunovariant adhesions, collectively calledP. falciparum erythrocyte membrane protein 1 (PfEMP1),to the erythrocyte’s surface, where they act as ligands forcytoadhesive events [1].In cells of higher eukaryotes, intracellular protein trans-port between the various organelles of the endomembranesystem and the plasma membrane is facilitated by vesi-cles. Many, if not all of the vesicles possess a protein coatat their cytosolic surface. The first coat described was theclathrin coat, which is characteristic of endosomal vesicu-lar transport from the plasma membrane to endosomes [2].Distinct from clathrin-coated vesicles are COPI (coat pro-tein I)-coated vesicles[3] that mediate transport steps withinthe Golgi stack and back to the endoplasmic reticulum (ER)[4,5]. The COPI coat was shown to consist of the regulatoryGTPase ARF and seven different protein subunits termed al-pha, beta, beta , gamma, delta, epsilon and zeta [6]. Antero-grade protein traffic from the ER to the Golgi is executed bythe COPII-coated vesicles[7,8]. COPII coats also require ac-tivity of a small GTPase, referred to here as Sar1-activatingcomplex. In addition, two combined complexes consistingof Sec13/31 and Sec23/24 have been described in COPIIprotein-coated vesicles in yeast[9,10], and the structure of

Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast.

Yeast produce two classes of secretory vesicles (SVs) that differ in both density and cargo protein content. In late-acting secretory mutants (e.g. snc1(ala43) and sec6-4), both low- (LDSV) and high-density (HDSV) classes of vesicles accumulate at restrictive temperatures. Here, we have found that disruptions in the genes encoding a dynamin-related protein (VPS1) or clathrin heavy chain (CHC1) abolish HDSV production, yielding LDSVs that contain all secreted cargos. Interestingly, disruption of the PEP12 gene, which encodes the t-SNARE that mediates all Golgi to pre-vacuolar compartment (PVC) transport, also abolishes HDSV production. In contrast, deletions in genes that selectively confer vacuolar hydrolase sorting to the PVC or protein transport to the vacuole (i.e. VPS34 and VAM3, respectively) have no effect. Thus, one branch of the secretory pathway in yeast involves an intermediate sorting compartment and has a specific requirement for clathrin and a dynamin-related protein in SV biogenesis.

Structural determinants of Ca2+ transport in the Arabidopsis H+/Ca2+ antiporter CAX1.

Ca(2+) levels in plants, fungi, and bacteria are controlled in part by H(+)/Ca(2+) exchangers; however, the relationship between primary sequence and biological activity of these transporters has not been reported. The Arabidopsis H(+)/cation exchangers, CAX1 and CAX2, were identified by their ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. CAX1 has a much higher capacity for Ca(2+) transport than CAX2. An Arabidopsis thaliana homolog of CAX1, CAX3, is 77% identical (93% similar) and, when expressed in yeast, localized to the vacuole but did not suppress yeast mutants defective in vacuolar Ca(2+) transport. Chimeric constructs and site-directed mutagenesis showed that CAX3 could suppress yeast vacuolar Ca(2+) transport mutants if a nine-amino acid region of CAX1 was inserted into CAX3 (CAX3-9). Biochemical analysis in yeast showed CAX3-9 had 36% of the H(+)/Ca(2+) exchange activity as compared with CAX1; however, CAX3-9 and CAX1 appear to differ in their transport of other ions. Exchanging the nine-amino acid region of CAX1 into CAX2 doubled yeast vacuolar Ca(2+) transport but did not appear to alter the transport of other ions. This nine-amino acid region is highly variable among the plant CAX-like transporters. These findings suggest that this region is involved in CAX-mediated Ca(2+) specificity.

PH and multidrug resistance.

Most chemotherapeutic drugs in use today are hydrophobic small molecules that are also typically either weakly basic, weakly acidic or charged. Thus, changes in the electrochemical parameters of tumour cell membranes have important effects on their transmembranous diffusion and cellular retention. Changes in these parameters can also modulate the function of immunological agents, and affect the signal transduction associated with induction of apoptosis. For these reasons, it is logical to propose that many (if not most) of the characteristics of multidrug-resistant (MDR) tumour cells could be due to perturbations in cellular ion transport. Indeed, many reports of altered ion transport in MDR cells can be found in the literature. Moreover, many studies suggest that P glycoprotein (Pgp) overexpression confers this altered ion transport, however, detailed physical-chemical analysis of this phenomenon has been confused by the complexity of the model systems devised to study Pgp. To help resolve this confusion, our laboratory has focused on a detailed characterization of 'pure' and stable Pgp transfectants unadulterated by the complications of chemotherapeutic drug exposure, various yeast strains and yeast vesicle preparations, and purified, reconstituted Pgp preparations. Recent data obtained with these model systems are summarized in this paper.

The role of GTP-binding protein activity in fast central synaptic transmission.

Guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are involved in exocytosis, endocytosis, and recycling of vesicles in yeast and mammalian secretory cells. However, little is known about their contribution to fast synaptic transmission. We loaded guanine nucleotide analogs directly into a giant nerve terminal in rat brainstem slices. Inhibition of G-protein activity had no effect on basal synaptic transmission, but augmented synaptic depression and significantly slowed recovery from depression. A nonhydrolyzable GTP analog blocked recovery of transmission from activity-dependent depression. Neither effect was accompanied by a change in presynaptic calcium currents. Thus, G proteins contribute to fast synaptic transmission by refilling synaptic vesicles depleted after massive exocytosis.

Effects of myosin-II antibody on actin-dependent vesicle transport in extracts of clam oocytes.

The movement of vesicles and organelles in cells has been shown to occur on both microtubules and actin filaments (1, 2). The transport of vesicles on microtubules permits movement over long distances in cells, while the short-range transport of vesicles on actin filaments produces the fine and precise movements required to position vesicles at membrane sites for capture, docking, or fusion (3, 4). The transport of vesicles on actin filaments requires vesicle-associated myosins, some of which have been identified. Myosin V, for example, transports endoplasmic reticulum (ER) vesicles (5-7) and synaptic vesicles (8) in neurons and is involved in the capture of melanosomes at the tips of mouse melanocytes (9). In addition, myosin V is involved in the transport of lysosomes in some cells (10), secretory vesicles in yeast (11), and melanosomes in Xenopus melanophores (12). Myosin II, also known as conventional myosin, is vesicleassociated in extracts of clam (Spisula solidissima) oocytes (4, 13). In addition, myosin II was reported to be associated with Golgi vesicles (14), but the antibody used in these studies cross-reacts with coatomer proteins on Golgi-derived vesicles (15-17). Therefore, the role of myosin II in vesicle transport remains controversial. In this study we performed antibody-inhibition experiments with extracts of clam oocytes to provide additional evidence that myosin II is a vesicle motor. Clam oocyte extracts are well characterized and have been used extensively to study cell cycle events including cyclin synthesis and degradation, as well as ubiquitin-mediated proteolysis of cyclins (18). Extracts were prepared from quiescent oocytes by the method of Rud rman et al., (18) with slight modifications. Fluorescence microscopy was used to view rhodamine-phalloidin stained actin filaments in the extracts, and AVEC-DIC microscopy was used to assay vesicle transport on actin filaments. Motile activity in extracts was determined by counting the number of vesicles moving per minute per field (v/m/f). Antibody-inhibition experiments were performed with an antibody made to oocyte yo in II. The myosin II in oocyte extracts was precipitated with ammonium sulfate, dissolved in buffer (20 mM Tris pH 7.2, 1 mM EDTA-K, 0.2 mM CaCl2, 1 mM DTT), and dialyzed overnight at 4?C. The pure fraction of the myosin II was prepared by running the solubilized fractions on SDS-gels and eluting the myosin II heavy chain band. The eluted protein was used f r antibody production in rabbit. The serum of the immunized rabbit was collected, and the polyclonal antibody (ap-205) was purified on protein A beads. The antibody (ap-205) recognized a single band at 205 kD on west n blots of oocyte extracts (Fig. 1A) and in myosin IIenr ched fractions (Fig. lB). The protein recognized by ap-205 wa also recognized by a myosin II antibody, M2.42 (Fig. 1A), produced to a peptide in the head domain of Acanthamoeba

Transmembrane helix 5 is critical for the high water permeability of aquaporin.

Aquaporin-2 (AQP2), a vasopressin-regulated water channel, plays a major role in urinary concentration. AQP2 and the major intrinsic protein (MIP) of lens fiber are highly homologous (58% amino acid identity) and share a topology of six transmembrane helices connected by five loops (loops A-E). Despite the similarities of these proteins, however, the water channel activity of AQP2 is much higher than that of MIP. To determine the site responsible for this gain of activity in AQP2, several parts of MIP were replaced with the corresponding parts of AQP2. When expressed in Xenopus oocytes, the osmotic water permeability (P(f)) of MIP and AQP2 was 48 and 245 x 10(-)(4) cm/s, respectively. Substitutions in loops B-D failed to increase P(f), whereas substitution of loop E significantly increased P(f) 1.5-fold. A similar increase in P(f) was observed with the substitution of the front half of loop E. P(f) measurements taken in a yeast vesicle expression system also confirmed that loop E had a complementary effect, whereas loops B-D did not. However, P(f) values of the loop E chimeras were only approximately 30% of that of AQP2. Simultaneous exchanges of loop E and a distal half of transmembrane helix 5 just proximal to loop E increased P(f) to the level of that of AQP2. Replacement of helix 5 alone stimulated P(f) 2.7-fold. Conversely, P(f) was decreased by 73% when helix 5 of AQP2 was replaced with that of MIP. Moreover, P(f) was stimulated 2.6- and 3.3-fold after helix 5 of AQP1 and AQP4 was spliced into MIP, respectively. Our findings suggested that the distal half of helix 5 is necessary for maximum water channel activity in AQP. We speculate that this portion contributes to the formation of the aqueous pore and the determination of the flux rate.

Direct Involvement of Phosphatidylinositol 4-Phosphate in Secretion in the Yeast Saccharomyces cerevisiae

The SEC14 gene encodes an essential phosphatidylinositol (PtdIns) transfer protein required for formation of Golgi-derived secretory vesicles in yeast. Suppressor mutations that rescue temperature-sensitive sec14 mutants provide an approach for determining the role of Sec14p in secretion. One suppressor, sac1-22, causes accumulation of PtdIns(4)P. SAC1 encodes a phosphatase that can hydrolyze PtdIns(4)P and certain other phosphoinositides. These findings suggest that PtdIns(4)P is limiting in sec14 cells and that elevation of PtdIns(4)P production can suppress the secretory defect. Correspondingly, we found that PtdIns(4)P levels were decreased significantly in sec14-3 mutants shifted to 37 degrees C and that sec14-3 cells could grow at an otherwise nonpermissive temperature (34 degrees C) when carrying a plasmid overexpressing PIK1, encoding one of two essential PtdIns 4-kinases. This effect is specific because overexpression of the other PtdIns 4-kinase gene (STT4) or a PtdIns 3-kinase gene (VPS34) did not rescue sec14-3 cells. To further address Pik1p function in secretion, two different pik1(ts) mutants were examined. Upon shift to restrictive temperature (37 degrees C), the PtdIns(4)P levels dropped by about 60% in both pik1(ts) strains within 1 h. During the same period, cells displayed a reduction (40-50%) in release of a secreted enzyme (invertase). However, similar treatment did not effect maturation of a vacuolar enzyme (carboxypeptidase Y). These findings indicate that, first, PtdIns(4)P limitation is a major contributing factor to the secretory defect in sec14 cells; second, Sec14p function is coupled to the action of Pik1p, and; third, PtdIns(4)P has an important role in the Golgi-to-plasma membrane stage of secretion.