Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway

Mutations of the PKD1 and PKD2 genes, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively, lead to autosomal dominant polycystic kidney disease. Interestingly, up-regulation or down-regulation of PKD1 or PKD2 leads to polycystic kidney disease in animal models, but their interrelations are not completely understood. We show here that full-length PC1 that interacts with PC2 via a C-terminal coiled-coil domain regulates PC2 expression in vivo and in vitro by down-regulating PC2 expression in a dose-dependent manner. Expression of the pathogenic mutant R4227X, which lacks the C-terminal coiled-coil domain, failed to down-regulate PC2 expression, suggesting that PC1-PC2 interaction is necessary for PC2 regulation. The proteasome and autophagy are two pathways that control protein degradation. Proteins that are not degraded by proteasomes precipitate in the cytoplasm and are transported via histone deacetylase 6 (HDAC6) toward the aggresomes. We found that HDAC6 binds to PC2 and that expression of full-length PC1 accelerates the transport of the HDAC6-PC2 complex toward aggresomes, whereas expression of the R4227X mutant fails to do so. Aggresomes are engulfed by autophagosomes, which then fuse with the lysosome for degradation; this process is also known as autophagy. We have now shown that PC1 overexpression leads to increased degradation of PC2 via autophagy. Interestingly, PC1 does not activate autophagy generally. Thus, we have now uncovered a new pathway suggesting that when PC1 is expressed, PC2 that is not bound to PC1 is directed to aggresomes and subsequently degraded via autophagy, a control mechanism that may play a role in autosomal dominant polycystic kidney disease pathogenesis.     

Modulation of the Plasma Membrane Ca2+ Pump

The plasma membrane calcium pump, which ejects Ca²⁺ from the cell, is regulated by calmodulin. In the absence of calmodulin, the pump is relatively inactive; binding of calmodulin to a specific domain stimulates its activity. Phosphorylation of the pump with protein kinase C or A may modify this regulation. Most of the regulatory functions of the enzyme are concentrated in a region at the carboxyl terminus. This region varies substantially between different isoforms of the pump, causing substantial differences in regulatory properties. The pump shares some motifs of the carboxyl terminus with otherwise unrelated proteins: The calmodulin-binding domain is a modified IQ motif (a motif which is present in myosins) and the last 3 residues of isoform 4b are a PDZ target domain. The pump is ubiquitous, with isoforms 1 and 4 of the pump being more widely distributed than 2 and 3. In some kinds of cells isoform 1 or 4 is missing, and is replaced by another isoform. Received: 26 January 1998/Revised: 6 April 1998     

Polycystin-1 activates and stabilizes the polycystin-2 channel

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent genetic disorder largely caused by mutations in the PKD1 and PKD2 genes that encode the transmembrane proteins polycystin-1 and -2, respectively. Both proteins appear to be involved in the regulation of cell growth and maturation, but the precise mechanisms are not yet well defined. Polycystin-2 has recently been shown to function as a Ca(2+)-permeable, non-selective cation channel. Polycystin-2 interacts through its cytoplasmic carboxyl-terminal region with a coiled-coil motif in the cytoplasmic tail of polycystin-1 (P1CC). The functional consequences of this interaction on its channel activity, however, are unknown. In this report, we show that P1CC enhanced the channel activity of polycystin-2. R742X, a disease-causing polycystin-2 mutant lacking the polycystin-1 interacting region, fails to respond to P1CC. Also, P1CC containing a disease-causing mutation in its coiled-coil motif loses its stimulatory effect on wild-type polycystin-2 channel activity. The modulation of polycystin-2 channel activity by polycystin-1 may be important for the various biological processes mediated by this molecular complex.     

RalA and RalB Proteins Are Ubiquitinated GTPases, and Ubiquitinated RalA Increases Lipid Raft Exposure at the Plasma Membrane

Ras GTPases signal by orchestrating a balance among several effector pathways, of which those driven by the GTPases RalA and RalB are essential to Ras oncogenic functions. RalA and RalB share the same effectors but support different aspects of oncogenesis. One example is the importance of active RalA in anchorage-independent growth and membrane raft trafficking. This study has shown a new post-translational modification of Ral GTPases: nondegradative ubiquitination. RalA (but not RalB) ubiquitination increases in anchorage-independent conditions in a caveolin-dependent manner and when lipid rafts are endocytosed. Forcing RalA mono-ubiquitination (by expressing a protein fusion consisting of ubiquitin fused N-terminally to RalA) leads to RalA enrichment at the plasma membrane and increases raft exposure. This study suggests the existence of an ubiquitination/de-ubiquitination cycle superimposed on the GDP/GTP cycle of RalA, involved in the regulation of RalA activity as well as in membrane raft trafficking.     

Whey Protein Hydrolysate Increases Translocation of GLUT-4 to the Plasma Membrane Independent of Insulin in Wistar Rats

Whey protein (WP) and whey protein hydrolysate (WPH) have the recognized capacity to increase glycogen stores. The objective of this study was to verify if consuming WP and WPH could also increase the concentration of the glucose transporters GLUT-1 and GLUT-4 in the plasma membrane (PM) of the muscle cells of sedentary and exercised animals. Forty-eight Wistar rats were divided into 6 groups (n = 8 per group), were treated and fed with experimental diets for 9 days as follows: a) control casein (CAS); b) WP; c) WPH; d) CAS exercised; e) WP exercised; and f) WPH exercised. After the experimental period, the animals were sacrificed, muscle GLUT-1 and GLUT-4, p85, Akt and phosphorylated Akt were analyzed by western blotting, and the glycogen, blood amino acids, insulin levels and biochemical health indicators were analyzed using standard methods. Consumption of WPH significantly increased the concentrations of GLUT-4 in the PM and glycogen, whereas the GLUT-1 and insulin levels and the health indicators showed no alterations. The physical exercise associated with consumption of WPH had favorable effects on glucose transport into muscle. These results should encourage new studies dealing with the potential of both WP and WPH for the treatment or prevention of type II diabetes, a disease in which there is reduced translocation of GLUT-4 to the plasma membrane.     

Purinergic P2Y1 Receptors Control Rapid Expression of Plasma Membrane Processes in Hippocampal Astrocytes

Astrocytes regulate neuronal activity and blood brain barrier through tiny plasma membrane branches or astrocytic processes (APs) making contact with synapses and brain vessels. Several transmitters released by astrocytes and exerting their action on several receptor classes expressed by astrocytes themselves influence their physiology. Here we found that APs are dynamically modulated by purines. In live imaging experiments carried out in rat hippocampal astrocytes, Gq-coupled P2Y1 receptor blockade with the selective antagonist MRS2179 (1 μM) or inhibition of its effector phospholipase C using U73122 (3 μM) produced APs retraction, while stimulation of the same receptor with the selective agonist 2MeSADP (100 μM) increased their number. Since astrocytes, among other transmitters, release ATP by several mechanisms including connexin hemichannels, we used the connexin hemichannel inhibitor carbenoxolone (100 μM) and APs retraction was observed. In our system we then measured expression or function of channels important for modulation of volume transmission and K+ buffering, aquaporin-4, and K+ inward rectifying (Kir) channels, respectively. Aquaporin-4 expression level did not change whereas, in whole-cell patch-clamp recordings performed to measure Kir current, we observed an increase in K+ current in all conditions where APs number was reduced. These data are supporting the idea of a dynamic modulation of astrocytic processes by purinergic signal, strengthening the role of purines in brain homeostasis.     

Biodiversity Increases the Productivity and Stability of Phytoplankton Communities

Global biodiversity losses provide an immediate impetus to elucidate the relationships between biodiversity, productivity and stability. In this study, we quantified the effects of species richness and species combination on the productivity and stability of phytoplankton communities subject to predation by a single rotifer species. We also tested one mechanism of the insurance hypothesis: whether large, slow-growing, potentially-defended cells would compensate for the loss of small, fast-growing, poorly-defended cells after predation. There were significant effects of species richness and species combination on the productivity, relative yield, and stability of phytoplankton cultures, but the relative importance of species richness and combination varied with the response variables. Species combination drove patterns of productivity, whereas species richness was more important for stability. Polycultures containing the most productive single species, Dunaliella, were consistently the most productive. Yet, the most species rich cultures were the most stable, having low temporal variability in measures of biomass. Polycultures recovered from short-term negative grazing effects, but this recovery was not due to the compensation of large, slow-growing cells for the loss of small, fast-growing cells. Instead, polyculture recovery was the result of reduced rotifer grazing rates and persisting small species within the polycultures. Therefore, although an insurance effect in polycultures was found, this effect was indirect and unrelated to grazing tolerance. We hypothesize that diverse phytoplankton assemblages interfered with efficient rotifer grazing and that this “interference effect” facilitated the recovery of the most productive species, Dunaliella. In summary, we demonstrate that both species composition and species richness are important in driving patterns of productivity and stability, respectively, and that stability in biodiverse communities can result from an alteration in consumer functioning. Our findings underscore the importance of predator-prey dynamics in determining the relationships between biodiversity, productivity and stability in producer communities.     

Immunochemical Analysis of the Temporal and Tissue-Specific Expression of an Avena sativa Plasma Membrane Determinant

An immunoglobulin Mk monoclonal (F8IVE9) antibody raised against oat (Avena sativa cv Garry) root homogenate has been produced and characterized. The predominant target bound by this antibody is a 62-kilodalton protein (p62) that is expressed in both oat root and oat shoot cells. Treatment of the oat antigen with periodate, or with recombinant N-glycanase, affects the F8IVE9 binding to the antigen, suggesting that the specific epitope for this monoclonal antibody involves a carbohydrate determinant. Levels of p62 present in cells of the oat root increase approximately twofold as the root tissue matures during the first 11 days postgermination. In contrast, levels of expression in shoot tissue remain relatively constant during the same period. The p62 antigen has been shown to be expressed at the plasma membrane by immunohistochemical means, by immunofluorescent labeling of protoplasts, and by enzyme-linked immunosorbent assay analysis of purified plasma membrane. The F8IVE9 antigenic target appears to be uniformly distributed through root tissue but is differentially expressed in specific sections of the shoot. F8IVE9 antibody also binds to antigens expressed in a number of other species, including clover, corn, pea, broccoli, mustard, and bean, and has been shown to bind to Samanea protoplast plasma membranes. This monoclonal antibody may prove to be useful for a variety of investigations, including an analysis of the specific patterns of cellular differentiation that occur during early morphogenesis, and the characterization of plasma membrane-associated elements in plants.     

质膜Ca2+-ATPase的结构与功能

质膜Ca2+-ATPase (PMCA)是P型ATPase家族的一员,在真核细胞中主要负责信号刺激后胞内高浓度Ca2+的清除扫尾工作,并对维持静息状态下较低Ca2+浓度起着重要的调节作用.PMCA的一级结构已被确定,拓扑学结构显示,它有10个跨膜区和3个胞浆功能区.它的4个编码基因可产生4种亚型(PMCA 1~4),这些亚型在功能与分布上存在差异.PMCA的活性可被钙调蛋白等多种因素调节,这与其结构特征息息相关.近年来,PMCA已被证实与脂筏结构有一定关联,它在信号传导和细胞凋亡中的作用也成为目前科学研究的焦点.本文主要对PMCA的结构、亚型和功能的研究现状进行综述.     

Identification of the Plasma Membrane Proteolipid Protein as a Constituent of Brain Coated Vesicles and Synaptic Plasma Membrane

We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.     

Identification of the Plasma Membrane Ca2+-ATPase and of Its Autoinhibitory Domain

The effect of controlled proteolysis on the plasma membrane (PM)Ca2+-ATPase was studied at the molecular level in PM purified from radish (Raphanus sativus L.) seedlings. Two new methods for labeling the PM Ca2+-ATPase are described. The PM Ca2+-ATPase can be selectively labeled by treatment with micromolar fluorescein isothiocyanate (FITC), a strong inhibitor of enzyme activity. Both inhibition of activity and FITC binding to the PM Ca2+-ATPase are suppressed by millimolar MgITP. The PM Ca2+-ATPase maintains the capability to bind calmodulin also after sodium dodecyl sulfate gel electrophoresis and blotting; therefore, it can be conveniently identified by 125l-calmodulin overlay in the presence of calcium. With both methods a molecular mass of 133 kD can be calculated for the PM Ca2+-ATPase. FITC-labeled PM Ca2+-ATPase co-migrates with the phosphorylated intermediate of the enzyme[mdash]labeled by incubation with [[gamma]-32P]GTP in the presence of calcium[mdash]on acidic sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Controlled trypsin treatment of purified PM determines a reduction of the molecular mass of the PM Ca2+-ATPase from 133 to 118 kD parallel to the increase of enzyme activity. Only the 133-kD but not the 118-kD PM Ca2+-ATPase binds calmodulin. These results indicate that trypsin removes from the PM Ca2+-ATPase an autoinhibitory domain that contains the calmodulin-binding domain of the enzyme.     

Expression of Plasma Membrane Water Channel Genes under Water Stress in Nicotiana excelsior

Deduced amino acid sequences encoded by the cDNAs related tothe MIP gene family from Nicotiana excelsior were characterized.Phylogenetic characterization of the products of correspondinggenes named NeMip1, NeMip2, and NeMip3 strongly suggested thatthey are water channel proteins localized in the plasma membrane.Organ specificity of the gene expression was examined in leaves,roots, and reproductive organs. NeMip1 was expressed in rootsand reproductive organs; however, it was hardly detectable inleaves. Two other genes, NeMip2 and NeMip3, were expressed inall of organs examined. mRNA accumulation from the genes wasinvestigated in leaves under salt- and drought-stresses. Theresults demonstrated that mRNA accumulation from all three genesincreased under salt- and drought-stresses within one day. However,they showed different accumulation patterns. In addition totheir up-reg-ulation under salt- and drought-stresses, dailychanges in NeMip2 and NeMip3 mRNA accumulation was observedunder unstressed conditions in leaves. (Received May 2, 1997; Accepted September 3, 1997)     

Liver-specific Deficiency of Serine Palmitoyltransferase Subunit 2 Decreases Plasma Sphingomyelin and Increases Apolipoprotein E Levels

Sphingomyelin (SM) is one of the major lipid components of plasma lipoproteins. Serine palmitoyltransferase (SPT) is the key enzyme in SM biosynthesis. Mice totally lacking in SPT are embryonic lethal. The liver is the major site for plasma lipoprotein biosynthesis, secretion, and degradation, and in this study we utilized a liver-specific knock-out approach for evaluating liver SPT activity and also its role in plasma SM and lipoprotein metabolism. We found that a deficiency of liver-specific Sptlc2 (a subunit of SPT) decreased liver SPT protein mass and activity by 95 and 92%, respectively, but had no effect on other tissues. Liver Sptlc2 deficiency decreased plasma SM levels (in both high density lipoprotein and non-high density lipoprotein fractions) by 36 and 35% (p < 0.01), respectively, and increased phosphatidylcholine levels by 19% (p < 0.05), thus increasing the phosphatidylcholine/SM ratio by 77% (p < 0.001), compared with controls. This deficiency also decreased SM levels in the liver by 38% (p < 0.01) and in the hepatocyte plasma membranes (based on a lysenin-mediated cell lysis assay). Liver-specific Sptlc2 deficiency significantly increased hepatocyte apoE secretion and thus increased plasma apoE levels 3.5-fold (p < 0.0001). Furthermore, plasma from Sptlc2 knock-out mice had a significantly stronger potential for promoting cholesterol efflux from macrophages than from wild-type mice (p < 0.01) because of a greater amount of apoE in the circulation. As a result of these findings, we believe that the ability to control liver SPT activity could result in regulation of lipoprotein metabolism and might have an impact on the development of atherosclerosis.Sphingomyelin (SM),² an amphipathic phospholipid located in the surface monolayer of all classes of plasma lipoproteins (LDL/very low density lipoprotein, 70–75%; HDL, 25–30%) (1), has significant effects on lipoprotein metabolism.A number of studies indicate that plasma SM levels influence the metabolism of apoB-containing lipoproteins. It has been reported that SM, but not cholesterol, significantly inhibits triglyceride lipolysis by lipoprotein lipase (2, 3). It has also been found that SM in lipoproteins delays remnant clearance by decreasing the binding of apoE to cell membrane receptors (4).Plasma SM levels also influence high density lipoprotein (HDL) metabolism. There have been reports that SM affects the structure of discoidal and spherical HDL (5). SM can inhibit lecithin-cholesterol acyltransferase by decreasing its binding to HDL (6). A negative correlation between the SM content of HDL and lecithin-cholesterol acyltransferase activity was observed in studies with proteoliposomes or reconstituted HDL (7). SM-rich recombinant HDL can inhibit scavenger receptor class B type I-mediated cholesterol ester-selective uptake in HepG2 cells (8).It is known that subendothelial retention and aggregation of atherogenic lipoproteins play an important role in atherogenesis (9). LDL extracted from human atherosclerotic lesions is highly enriched in SM than in plasma LDL (10–13). LDL retained in atherosclerotic lesions is acted on by an arterial wall sphingomyelinase, which promotes aggregation by converting SM to ceramide (10–12). Sphingomyelinase deficiency diminishes lipoprotein retention within early atheromata and prevents lesion progression (14). The ratio of SM to PC is increased 5-fold in very low density lipoprotein from hypercholesterolemic rabbits (15). ApoE knock-out (KO) mice are a well known atherogenic model. It has been shown that plasma SM levels in these mice are 4-fold higher than in WT animals (16), and this may contribute to increased atherosclerosis (17, 18). It has also been shown that in humans, higher plasma SM levels and SM/PC ratios are independent risk factors for coronary heart disease (19, 20). All these data suggest that plasma SM plays a critical role in the development of atherosclerosis.The interaction of SM, cholesterol, and glycosphingolipids drives the formation of plasma membrane rafts (21). These rafts, formed in the Golgi apparatus, are targeted to the plasma membranes, where they are thought to exist as islands within the sea of bulk membrane. Although there is disagreement as to their content, rafts are considered in most reports to include about 3500 lipid molecules and 30 proteins (22). As much as 65% of the total cellular SM is located in these rafts (23). Manipulation of membrane SM levels by sphingomyelinase can alter lipid raft composition, thus modifying cell function. For example, cholesterol efflux from macrophage foam cells, a key step in reverse cholesterol transport, requires trafficking of cholesterol from intracellular sites to the plasma membranes. Sphingomyelinase deficiency decreases cholesterol efflux through promoting cholesterol sequestration by SM (24).The biochemical synthesis of SM occurs through the actions of serine palmitoyl-CoA transferase (SPT), 3-ketosphinganine reductase, ceramide synthase, dihydroceramide desaturase, and sphingomyelin synthase. Located in the endoplasmic reticulum membranes, SPT is the rate-limiting enzyme in the pathway (25). Mammalian SPT contains two subunits, Sptlc1 and Sptlc2, encoding 53- and 63-kDa proteins, respectively (26, 27). Data from in vivo and in vitro studies suggest that each subunit is stabilized by forming a dimer (or possibly larger multimer) with the other (28). Another subunit, Sptlc3, has been reported (29), and it is important that its functions be further characterized.Mice totally lacking Sptlc1 or Sptlc2 are embryonic lethal (30). Because the liver is the major site for plasma lipoprotein biosynthesis, secretion, and degradation, we utilized a liver-specific knock-out approach for evaluating liver SPT activity and its role in plasma SM and lipoprotein metabolism. We found that Sptlc2 deficiency in the liver decreases plasma SM and increases apoE levels.     

Expression and Cellular Distribution Pattern of Plasma Membrane Calcium Pump Isoforms in Rat Pancreatic Islets

This work is aimed at identifying the presence and cellular distribution pattern of plasma membrane calcium pump (PMCA) isoforms in normal rat pancreatic islet. Microsomal fractions of isolated islets and exocrine tissue were analyzed to detect different PMCA isoforms. The cellular distribution pattern of these PMCAs in the islets was also studied in fixed pancreas sections incubated with antibodies against PMCAs and insulin. Antibody 5F10, which reacts with all PMCA variants, showed multiple bands in the blots in the 127-134 kDa region, indicating the presence of several isoforms. Microsomes also reacted positively with specific antibodies for individual PMCA isoforms, generating a band of the expected size. Antibody 5F10 immunocytochemically labeled the plasma cell membrane of both b- and non-b-cells, but predominantly the former. All islet cells were also labeled with antibodies against isoforms 1 and 4, while the antibody reacting with isoform 3 labeled exclusively b-cells. A few b- and non-b-cells were positively labeled with the antibody reacting with PMCA b variant. Negative results were obtained with the antibody against isoform 2. Further studies, together with previous reports on the modulatory effect of insulin secretagogues and blockers upon PMCA activity, may provide evidence of the importance of this particular PMCA expression for islet function under normal and pathological conditions.     

Thermal Stability of the Plasma Membrane Calcium Pump. Quantitative Analysis of Its Dependence on Lipid-Protein Interactions

Thermal stability of plasma membrane Ca²⁺ pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 ± 12 kJ/mol in the range 33–45°C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca²⁺ pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K _LD ) showing that those phospholipids with higher affinity provide greater stability to the Ca²⁺ pump. We developed a method for directly measure K _LD by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K _LD values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity. Received: 5 August 1999/Revised: 20 October 1999     

Polycystin-2 takes different routes to the somatic and ciliary plasma membrane

Polycystin-2 (also called TRPP2), an integral membrane protein mutated in patients with cystic kidney disease, is located in the primary cilium where it is thought to transmit mechanical stimuli into the cell interior. After studying a series of polycystin-2 deletion mutants we identified two amino acids in loop 4 that were essential for the trafficking of polycystin-2 to the somatic (nonciliary) plasma membrane. However, polycystin-2 mutant proteins in which these two residues were replaced by alanine were still sorted into the cilium, thus indicating that the trafficking routes to the somatic and ciliary plasma membrane compartments are distinct. We also observed that the introduction of dominant-negative Sar1 mutant proteins and treatment of cells with brefeldin A prevented the transport into the ciliary plasma membrane compartment, whereas metabolic labeling experiments, light microscopical imaging, and high-resolution electron microscopy revealed that full-length polycystin-2 did not traverse the Golgi apparatus on its way to the cilium. These data argue that the transport of polycystin-2 to the ciliary and to the somatic plasma membrane compartments originates in a COPII-dependent fashion at the endoplasmic reticulum, that polycystin-2 reaches the cis side of the Golgi apparatus in either case, but that the trafficking to the somatic plasma membrane goes through the Golgi apparatus whereas transport vesicles to the cilium leave the Golgi apparatus at the cis compartment. Such an interpretation is supported by the finding that mycophenolic acid treatment resulted in the colocalization of polycystin-2 with GM130, a marker of the cis-Golgi apparatus. Remarkably, we also observed that wild-type Smoothened, an integral membrane protein involved in hedgehog signaling that under resting conditions resides in the somatic plasma membrane, passed through the Golgi apparatus, but the M2 mutant of Smoothened, which is constitutively located in the ciliary but not in the somatic plasma membrane, does not. Finally, a dominant-negative form of Rab8a, a BBSome-associated monomeric GTPase, prevented the delivery of polycystin-2 to the primary cilium whereas a dominant-negative form of Rab23 showed no inhibitory effect, which is consistent with the view that the ciliary trafficking of polycystin-2 is regulated by the BBSome.     

Microsporidian Spore Discharge and the Transfer of Polaroplast Organelle Membrane into Plasma Membrane

Electron microscopic examinations of Glugea hertwigi and Spraguea lophii spores indicated the presence of a single plasma membrane; however, this membrane remained in the spore during the discharge of the sporoplasm from the spore. Although discharged spores retained the old plasma membrane, the extruded sporoplasms acquired a new plasma membrane. In order to determine where the new plasma membrane came from, we used two fluorescent probes with membrane affinities. The markers were tested on unfired and discharged spores. The probe, N-phenyl-1-naphthylamine (NPN), labeled the polaroplast membrane in addition to the apolar groups in the posterior vacuoles of unfired spores. After spore discharge, NPN label disappeared from the spore ghosts except for a slight fluorescence on residual plasma membranes. Much of the NPN-labeled membrane reappeared after spore discharge on the outer envelope of discharged sporoplasms. The probe chlorotetracycline (CTC) labeled calcium-associated membranes of spore polaroplasts. During spore discharge, the CTC fluorescence shifted from the polaroplast organelle of unfired spores to the outer envelope of discharged sporoplasms. These results indicate that the polaroplast organelle may provide the new plasma membrane for discharged microsporidian sporoplasms.