Regulation Of Membrane Transport Research Articles

Cross-linking of glycosphingolipids at the plasma membrane: consequences for intracellular signaling and traffic.

Glycosphingolipids (GSLs) are predominantly found in the outer leaflet of the plasma membrane, where they play a role in important processes such as cell adhesion, migration and signaling. However, by which mechanisms GSLs regulate these processes remains elusive. In this study, we therefore took advantage of the fact that some GSLs also serve as receptors for certain protein toxins, which rely on receptor binding for internalization and intoxication. Here, we demonstrate that Shiga and cholera toxins, which both possess multivalent GSL-binding capacity, induce dissociation of the cytosolic cPLA2α-AnxA1 complex in HeLa and HMEC-1 cells. The dissociation is mediated through an increase in cytosolic calcium levels and activation of the tyrosine kinase Syk. Ricin, a protein toxin that does not cross-link surface molecules, has no effect on the same complex. Importantly, we find that antibody-mediated cross-linking of Gb3 and GM1, the GSL receptors for Shiga and cholera toxin, respectively, also induces dissociation. These data demonstrate that cross-linking of GSLs at the plasma membrane mediates the intracellular signaling events resulting in dissociation of the complex. After dissociation, cPLA2α and AnxA1 are translocated to intracellular membranes where they are known to function in regulating membrane transport processes. In conclusion, we have characterized a novel mechanism for cell surface-induced initiation of intracellular signaling and transport events.

Abstract 4415: Tyrosine phosphorylation is essential for OCT2 function and a target of inhibition by TKIs

Abstract Cisplatin and oxaliplatin are among the most widely used anticancer drugs in both children and adults. The clinical use of these agents is associated with dose-limiting damage to kidneys (nephrotoxicity) and peripheral nerves (neurotoxicity). Mechanistic understanding of the underlying etiology remains incomplete and preventive strategies are currently unavailable. We previously reported that cisplatin nephrotoxicity (Filipski et al, CPT 2009) and oxaliplatin neurotoxicity (Sprowl et al, PNAS 2013) are dependent on organic cation transporter-mediated uptake mechanisms and can be reduced by genetic or pharmacological inhibition of OCT2. With a goal to identify small molecule OCT2 inhibitors, that could be used to prevent these toxicities, we carried out a high-throughput chemical library screen. Multiple tyrosine kinase inhibitors (TKIs), including dasatinib and vandetanib, were identified as potent OCT2 inhibitors. Further studies showed that TKIs inhibit OCT2 through a non-competitive mechanism raising the possibility that tyrosine phosphorylation by an unknown TKI-sensitive protein kinase(s) may be essential for OCT2 function. Using in vitro cell culture models and proteomics, site-directed mutagenesis and functional assays we found that OCT2 is indeed tyrosine phosphorylated and this phosphorylation is essential for OCT2 function and sensitive to inhibition by TKIs. A subsequent RNAi kinase library screen identified the Src-family member YES1 as the OCT2-phosphorylating kinase. This was further supported by experiments where a TKI-resistant YES1 mutant was able to rescue OCT2 inhibition by dasatinib. Non-TKI inhibitors of YES1 were also found to suppress OCT2 function. Importantly, tyrosine phosphorylation of OCT2 was observed in vivo, in murine kidneys and dorsal root ganglions, major sites of OCT2 expression and toxicities of platinum-based drugs. siRNA mediated in vivo knockdown of YES1 reduced OCT2 phosphorylation and suppressed its function in the kidneys. Treatment with the YES1 targeting TKI, Dasatinib, suppressed OCT2 phosphorylation in dorsal root ganglions, reduced oxaliplatin uptake and significantly prevented oxaliplatin induced neurotoxicity in mice models. These findings have not only provided new mechanistic insights into the regulation of membrane transporters; but have also provided a rationale for the translational development of new targeted interventions using TKI pulse-exposure to mitigate the debilitating side effects associated with platinum-based chemotherapeutics. Note: This abstract was not presented at the meeting. Citation Format: Navjotsingh Pabla, Jason A. Sprowl, Su Sien Ong, Alice A. Gibson, Guoqing Du, Wenwei Lin, Shuiying Hu, Lie Li, Taosheng Chen, Alex Sparreboom. Tyrosine phosphorylation is essential for OCT2 function and a target of inhibition by TKIs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4415. doi:10.1158/1538-7445.AM2015-4415

BSND and ATP6V1G3: Novel Immunohistochemical Markers for Chromophobe Renal Cell Carcinoma.

Differentiating between chromophobe renal cell carcinoma (RCC) and other RCC subtypes can be problematic using routine light microscopy. This study aimed to identify novel immunohistochemical markers useful for a differential diagnosis between chromophobe RCC and other RCC subtypes.We selected 3 genes (including BSND and ATP6V1G3) that showed specific transcriptional expression in chromophobe RCC using expression data (n = 783) from The Cancer Genome Atlas (TCGA) database. A subsequent immunohistochemical examination of 186 RCCs obtained in our patient series resulted in a strong diffuse positivity of BSND and ATP6V1G3 proteins (both of which are involved in the regulation of membrane transport) in all the chromophobe RCC specimens (23/23 cases, 100%) but not in the clear cell RCC specimens (0/153 cases, 0%) or the papillary RCC specimens (0/10 cases, 0%). BSND and ATP6V1G3 protein expressions were also detected in renal oncocytoma (13/14 cases, 92.9%) and in the distal nephron, including the collecting duct, in the normal kidney. A computational analysis of TCGA data suggested that DNA methylation was involved in the differential expression pattern of both genes among RCC subtypes. Finally, an immunohistochemical analysis showed lung carcinomas were negative (0/85 cases, 0%) for the expression of both proteins.These results suggest that BSND and ATP6V1G3 are excellent novel immunohistochemical markers for differentiating between chromophobe RCC and other subtypes of RCC, including clear cell and papillary RCCs.

Plant salt tolerance: adaptations in halophytes.

Most of the water on Earth is seawater, each kilogram of which contains about 35 g of salts, and yet most plants cannot grow in this solution; less than 0·2% of species can develop and reproduce with repeated exposure to seawater. These 'extremophiles' are called halophytes. Improved knowledge of halophytes is of importance to understanding our natural world and to enable the use of some of these fascinating plants in land re-vegetation, as forages for livestock, and to develop salt-tolerant crops. In this Preface to a Special Issue on halophytes and saline adaptations, the evolution of salt tolerance in halophytes, their life-history traits and progress in understanding the molecular, biochemical and physiological mechanisms contributing to salt tolerance are summarized. In particular, cellular processes that underpin the ability of halophytes to tolerate high tissue concentrations of Na+ and Cl−, including regulation of membrane transport, their ability to synthesize compatible solutes and to deal with reactive oxygen species, are highlighted. Interacting stress factors in addition to salinity, such as heavy metals and flooding, are also topics gaining increased attention in the search to understand the biology of halophytes. Halophytes will play increasingly important roles as models for understanding plant salt tolerance, as genetic resources contributing towards the goal of improvement of salt tolerance in some crops, for re-vegetation of saline lands, and as 'niche crops' in their own right for landscapes with saline soils.

The Rho-specific GAP protein DLC3 coordinates endocytic membrane trafficking

Membrane trafficking is known to be coordinated by small GTPases, but the identity of their regulators, the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that ensure balanced GTPase activation at different subcellular sites is largely elusive. Here, we show in living cells that deleted in liver cancer 3 (DLC3, also known as STARD8) is a functional Rho-specific GAP protein, the loss of which enhances perinuclear RhoA activity. DLC3 is recruited to Rab8-positive membrane tubules and is required for the integrity of the Rab8 and Golgi compartments. Depletion of DLC3 impairs the transport of internalized transferrin to the endocytic recycling compartment (ERC), which is restored by the simultaneous downregulation of RhoA and RhoB. We further demonstrate that DLC3 loss interferes with epidermal growth factor receptor (EGFR) degradation associated with prolonged receptor signaling. Taken together, these findings identify DLC3 as a novel component of the endocytic trafficking machinery, wherein it maintains organelle integrity and regulates membrane transport through the control of Rho activity.

Role of resveratrol in regulation of membrane transporters and integrity of human erythrocytes

An altered ion homeostasis due to impaired membrane transporters is known to be involved in the pathophysiology of many chronic diseases. Resveratrol, a phytoalexin, has been reported to elicit pleiotropic health-promoting effects, however, the mechanism(s) which underlie these effects remain speculative. The present study investigate the modulatory role of resveratrol on erythrocyte membrane Ca2+ATPase (PMCA pump), Na+/K+-ATPase (NKA pump), and Na+/H+ exchanger (NHE) in control and experimental-oxidative stress conditions. Results suggest that resveratrol is a potent modulator of membrane transporters evidenced by stimulation of PMCA and NKA pumps and down-regulation of NHE. The observed effects on membrane transporters correlated with susceptibility of erythrocyte membrane to oxidative damage. The findings provide an insight into the role of membrane transporters and their involvement in the health beneficial effects of resveratrol.

Src Family Protein Tyrosine Kinase Regulates the Basolateral K Channel in the Distal Convoluted Tubule (DCT) by Phosphorylation of KCNJ10 Protein

The loss of function of the basolateral K channels in the distal nephron causes electrolyte imbalance. The aim of this study is to examine the role of Src family protein tyrosine kinase (SFK) in regulating K channels in the basolateral membrane of the mouse initial distal convoluted tubule (DCT1). Single-channel recordings confirmed that the 40-picosiemen (pS) K channel was the only type of K channel in the basolateral membrane of DCT1. The suppression of SFK reversibly inhibited the basolateral 40-pS K channel activity in cell-attached patches and decreased the Ba(2+)-sensitive whole-cell K currents in DCT1. Inhibition of SFK also shifted the K reversal potential from -65 to -43 mV, suggesting a role of SFK in determining the membrane potential in DCT1. Western blot analysis showed that KCNJ10 (Kir4.1), a key component of the basolateral 40-pS K channel in DCT1, was a tyrosine-phosphorylated protein. LC/MS analysis further confirmed that SFK phosphorylated KCNJ10 at Tyr(8) and Tyr(9). The single-channel recording detected the activity of a 19-pS K channel in KCNJ10-transfected HEK293T cells and a 40-pS K channel in the cells transfected with KCNJ10+KCNJ16 (Kir.5.1) that form a heterotetramer in the basolateral membrane of the DCT. Mutation of Tyr(9) did not alter the channel conductance of the homotetramer and heterotetramer. However, it decreased the whole-cell K currents, the probability of finding K channels, and surface expression of KCNJ10 in comparison to WT KCNJ10. We conclude that SFK stimulates the basolateral K channel activity in DCT1, at least partially, by phosphorylating Tyr(9) on KCNJ10. We speculate that the modulation of tyrosine phosphorylation of KCNJ10 should play a role in regulating membrane transport function in DCT1.

Role of cytochrome P450 epoxygenase in regulating renal membrane transport and hypertension

Cytochrome P450 (CYP)-epoxygenase is highly expressed in the kidney and its metabolism of arachidonic acid plays important roles in regulating renal Na transport and in modulating vasoactivity in the kidney. In the past several years, progress has been made not only in characterizing the specific CYP-epoxygenases responsible for the regulation of membrane transport and vasoactivity in the kidney but also in exploring the mechanism by which they regulate renal Na transport and vasodilation of preglomerular arterioles. This review summarizes and updates recent progress in this area of research. CYP-epoxygenase metabolites of arachidonic acid inhibit epithelial Na channel (ENaC) in the cortical collecting duct (CCD), and 11,12-epoxyeicosatrienoic acid (11,12-EET) is mainly responsible for mediating the inhibitory effect on ENaC. Downregulation of CYP2C44 abolishes arachidonic acid mediated inhibition of ENaC and increases ENaC activity. In addition, 11,12-EET stimulates Ca-activated big conductance K channels in the CCD and afferent arterioles smooth muscles. Activation of big conductance K channels by 11,12-EET is responsible for EET-induced vasodilation in preglomerular arterioles. 11,12-EET-induced vasodilation is absent in preglomerular arterioles pretreated with okadaic acid. CYP-epoxygenase mediated suppression of renal Na transport is partially achieved by inhibition of ENaC activity in the CCD and CYP2C44-derived EETs are responsible for inhibition of ENaC. Stimulation of serine/threonine protein phosphatase 2A (PP2A) contributes to 11,12-EET-induced activation of big conductance K channels and vasodilation in preglomerular arterioles.

Molecular Characterization of Alkaline Phosphatase and PLAP in the Germinal Epithelium of Pb and Se+Zn Treated Adult Sprague Dawley Rats

Aim: The role of alkaline phosphatase in cell metabolism and regulation is essential and cuts across cell proliferation, division, cell death, membrane transport and DNA cleavage. This study addresses the role and importance of alkaline phosphates (ALP) and Placenta Alkaline phosphatase (PLAP) in the cells of germinal epithelium of male Rat testes following induced lead (Pb) toxicity and rejuvenation by Selenium (Se) and Zinc (Zn) treatment. Method: Adult Sprague-Dawley Rats (males) were divided into 4 groups of 15 animals each and were treated as thus; Group 1: Normal saline and serves as the control, Group 2: 100 mg/Kg BW of Pb only, Group 3: 100 mg/Kg BW of Pb and 2.25 mg/Kg each of Se and Zn and group 4 received 2.25 mg.Kg each of Se and Zn only. The duration of treatment was 56 days following which the animals were sacrificed by the 57th day and testes fixed in Bouin’s fluid. Result: Pb induced toxicity could be apoptotic involving ROS activation of NO-dependent apoptotic pathway or necrotic involving a wide range damage by ROS induced lipid peroxidation. Selenium and zinc treatment ameliorated the damage induced by lead toxicity in the germinal epithelium. Conclusion: ALP is involved in intrinsic regulation of DNA cleavage in apoptosis by functioning similar to endonucleases and also regulates membrane transport in perioxidated biomembranes. PLAP expression is minimal as tumorgenesis was not recorded, although Pb treatment showed signs of epithelium of endothelial cancer signalling, which was ameliorated by Se+Zn treatment.

Rab40b regulates MMP2 and MMP9 trafficking during invadopodia formation and breast cancer cell invasion

Invadopodia-dependent degradation of the basement membrane plays a major role during metastasis of breast cancer cells. Basement membrane degradation is mediated by targeted secretion of various matrix metalloproteinases (MMPs). Specifically, MMP2 and MMP9 (MMP2/9) possess the ability to hydrolyze components of the basement membrane and regulate various aspects of tumor growth and metastasis. However, the membrane transport machinery that mediates targeting of MMP2/9 to the invadopodia during cancer cell invasion remains to be defined. Because Rab GTPases are key regulators of membrane transport, we screened a human Rab siRNA library and identified Rab40b GTPase as a protein required for secretion of MMP2/9. We also have shown that Rab40b functions during at least two distinct steps of MMP2/9 transport. Here, we demonstrate that Rab40b is required for MMP2/9 sorting into VAMP4-containing secretory vesicles. We also show that Rab40b regulates transport of MMP2/9 secretory vesicles during invadopodia formation and is required for invadopodia-dependent extracellular matrix degradation. Finally, we demonstrate that Rab40b is also required for breast cancer cell invasion in vitro. On the basis of these findings, we propose that Rab40b mediates trafficking of MMP2/9 during invadopodia formation and metastasis of breast cancer cells.

Arl5b is a Golgi-localised small G protein involved in the regulation of retrograde transport

Regulation of membrane transport is controlled by small G proteins, which include members of the Rab and Arf families. Whereas the role of the classic Arf family members are well characterized, many of the Arf-like proteins (Arls) remain poorly defined. Here we show that Arl5a and Arl5b are localised to the trans-Golgi in mammalian cells, and furthermore have identified a role for Arl5b in the regulation of retrograde membrane transport from endosomes to the trans-Golgi network (TGN). The constitutively active Arl5b (Q70L)-GFP mutant was localised efficiently to the Golgi in HeLa cells whereas the dominant-negative Arl5b (T30N)-GFP mutant was dispersed throughout the cytoplasm and resulted in perturbation of the Golgi apparatus. Stable HeLa cells expressing GFP-tagged Arl5b (Q70L) showed an increased rate of endosome-to-Golgi transport of the membrane cargo TGN38 compared with control HeLa cells. Depletion of Arl5b by RNAi resulted in an alteration in the intracellular distribution of mannose-6-phosphate receptor, and significantly reduced the endosome-to-TGN transport of the membrane cargo TGN38 and of Shiga toxin, but had no affect on the anterograde transport of the cargo E-cadherin. Collectively these results suggest that Arl5b is a TGN-localised small G protein that plays a key role in regulating transport along the endosome-TGN pathway.

Subversion of membrane transport pathways by vacuolar pathogens

Mammalian phagocytes control bacterial infections effectively through phagocytosis, the process by which particles engulfed at the cell surface are transported to lysosomes for destruction. However, intracellular pathogens have evolved mechanisms to avoid this fate. Many bacterial pathogens use specialized secretion systems to deliver proteins into host cells that subvert signaling pathways controlling membrane transport. These bacterial effectors modulate the function of proteins that regulate membrane transport and alter the phospholipid content of membranes. Elucidating the biochemical function of these effectors has provided a greater understanding of how bacteria control membrane transport to create a replicative niche within the host and provided insight into the regulation of membrane transport in eukaryotic cells.

Synthesis, Maturation, and Trafficking of Human Na+-Dicarboxylate Cotransporter NaDC1 Requires the Chaperone Activity of Cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na(+)-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

Endosome-to-Golgi transport pathways in physiological processes.

The trans-Golgi network (TGN) is a major traffic hub of the cell, as it regulates membrane transport in the secretory pathway as well as receiving protein cargo by retrograde transport from endocytic compartments. Retrograde transport between endosomes and the TGN is essential for the recycling of membrane proteins which regulate a range of cellular and development functions. In addition, retrograde transport pathways are exploited by many bacterial toxins to mediate cytotoxicity and by some viral proteins to promote pathogenicity. Recent advances using a range of molecular cell biological strategies have identified multiple retrograde transport pathways each regulated by a distinct set of molecular machinery. Here we review recent advances in this field and highlight the importance of these transport pathways in a range of physiological processes.

Alterations in the proteome of the NHERF1 knockout mouse jejunal brush border membrane vesicles

Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCβ3, E-cadherin, p120, β-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.

Crystallization of the Membrane-Associated Annexin B1: Roles of Additive Screen, Dynamic Light Scattering, and Bioactivity Assay

Annexin B1 (AnxB1) is a calcium-dependent phospholipid binding protein from Taenia solium cysticercus and has been reported to possess anticoagulant activity, to inhibit phospholipase A2, and to regulate membrane transport. Native AnxB1 and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. The results of dynamic light scattering analysis showed that Hepes buffer combined with low concentration salts (NaCl or CaCl2) was beneficial for preventing aggregation and for AnxB1 stabilization in the storage. After the additive screen, crystals have been yielded in the presence of guanidine hydrochloride (Gn-HCl). We determined that a low concentration of Gn-HCl significantly delayed clotting time and increased anticoagulant activity. Analysis of the crystal showed that in the presence of Gn-HCl, AnxB1 crystallizes in orthorhombic space group, which is modified from the cubic space group for crystals grown in the absence of Gn-HCl. A high quality data set (at 1.9 A) has been co...

RAB6C Is a Retrogene that Encodes a Centrosomal Protein Involved in Cell Cycle Progression

Rab-GTPases are key regulators of membrane transport, and growing evidence indicates that their expression levels are altered in certain human malignancies, including cancer. Rab6C, a newly identified Rab6 subfamily member, has attracted recent attention because its reduced expression might confer a selective advantage to drug-resistant breast cancer cells. Here, we report that RAB6C is a primate-specific retrogene derived from a RAB6A′ transcript. RAB6C is transcribed in a limited number of human tissues including brain, testis, prostate, and breast. Endogenous Rab6C is considerably less abundant and has a much shorter half-life than Rab6A′. Comparison of the GTP-binding motifs of Rab6C and Rab6A′, homology modeling, and GTP-blot overlay assays indicate that amino acid changes in Rab6C have greatly reduced its GTP-binding affinity. Instead, the noncanonical GTP-binding domain of Rab6C mediates localization of the protein to the centrosome. Overexpression of Rab6C results in G1 arrest, and its specific depletion generates tetraploid cells with supernumerary centrosomes, revealing a role of Rab6C in events related to the centrosome and cell cycle progression. Thus, RAB6C is a rare example of a recently emerged retrogene that has acquired the status of a new gene, encoding a functional protein with altered characteristics compared to Rab6A′.

Identification of the Penta-EF-hand Protein ALG-2 as a Ca2+-dependent Interactor of Mucolipin-1

Loss of function mutations in mucolipin-1 (MCOLN1) have been linked to mucolipidosis type IV (MLIV), a recessive lysosomal storage disease characterized by severe neurological and ophthalmological abnormalities. MCOLN1 is an ion channel that regulates membrane transport along the endolysosomal pathway. It has been suggested that MCOLN1 participates in several Ca(2+)-dependent processes, including fusion of lysosomes with the plasma membrane, fusion of late endosomes and autophagosomes with lysosomes, and lysosomal biogenesis. Here, we searched for proteins that interact with MCOLN1 in a Ca(2+)-dependent manner. We found that the penta-EF-hand protein ALG-2 binds to the NH-terminal cytosolic tail of MCOLN1. The interaction is direct, strictly dependent on Ca(2+), and mediated by a patch of charged and hydrophobic residues located between MCOLN1 residues 37 and 49. We further show that MCOLN1 and ALG-2 co-localize to enlarged endosomes induced by overexpression of an ATPase-defective dominant-negative form of Vps4B (Vps4B(E235Q)). In agreement with the proposed role of MCOLN1 in the regulation of fusion/fission events, we found that overexpression of MCOLN1 caused accumulation of enlarged, aberrant endosomes that contain both early and late endosome markers. Interestingly, aggregation of abnormal endosomes was greatly reduced when the ALG-2-binding domain in MCOLN1 was mutated, suggesting that ALG-2 regulates MCOLN1 function. Overall, our data provide new insight into the molecular mechanisms that regulate MCOLN1 activity. We propose that ALG-2 acts as a Ca(2+) sensor that modulates the function of MCOLN1 along the late endosomal-lysosomal pathway.

Analysis and identification of ADP-ribosylated proteins of Streptomyces coelicolor M145

Mono-ADP-ribosylation is the enzymatic transfer of ADP-ribose from NAD(+) to acceptor proteins catalyzed by ADP-ribosyltransferases. Using m-aminophenylboronate affinity chromatography, 2D-gel electrophoresis, in-gel digestion and MALDI-TOF analysis we have identified eight in vitro ADP-ribosylated proteins in Streptomyces coelicolor, which can be classified into three categories: (i) secreted proteins; (ii) metabolic enzymes using NAD(+)/NADH or NADP(+)/NADPH as coenzymes; and (iii) other proteins. The secreted proteins could be classified into two functional categories: SCO2008 and SC05477 encode members of the family of periplasmic extracellular solute-binding proteins, and SCO6108 and SC01968 are secreted hydrolases. Dehydrogenases are encoded by SC04824 and SC04771. The other targets are GlnA (glutamine synthetase I., SC02198) and SpaA (starvation-sensing protein encoded by SC07629). SCO2008 protein and GlnA had been identified as ADP-ribosylated proteins in previous studies. With these results we provided experimental support for a previous suggestion that ADP-ribosylation may regulate membrane transport and localization of periplasmic proteins. Since ADP-ribosylation results in inactivation of the target protein, ADP-ribosylation of dehydrogenases might modulate crucial primary metabolic pathways in Streptomyces. Several of the proteins identified here could provide a strong connection between protein ADP-ribosylation and the regulation of morphological differentiation in S. coelicolor.

Proteins, membranes and cells: the structure–function nexus—ASB 2008

While it is a common misconception that the laboratory is ‘a quiet garden of Eden’ for the introverted investigator it is nevertheless a stark diVerence from the hub-bub and buzz of a scientiWc meeting. The latter was also the case with the 32nd Annual Meeting of the Australian Society for Biophysics that took place at the Australian National University in Canberra from September 28th to October 1st, 2008. In an era when many national and international conferences are arranged by professional meeting organisers, held in purpose-built convention centres, and employ multiple parallel sessions, it is refreshing to attend a smaller conference where all the lectures are held in a single auditorium. This is particularly true when such a conference encompasses the breadth of research covered by Biophysics, as it aVords the opportunity to catch up with a lot of interesting ideas that one usually doesn’t have the time to think about in today’s busy schedules. The themes covered at the meeting that are reXected in the articles in this special edition, included Ion Channels, Fundamental Issues and Regulation of Membrane Transport, Protein Structure and Dynamics, Muscle Biophysics, Biophysical Characterisation Techniques, Plant Biophysics and Photosynthesis, Membrane Biophysics, General Biophysics, Cellular & Molecular Biophysics and Bioengineering, Biomolecules, Biomaterials and Biomimetics. The Wrst series of articles in this special issue are based on presentations given at a session held in memory of Professor Alexander (“Alex”) B. Hope and his work; Alex died in July 2008. He was one of the founders of biophysical research in Australia, so we were very fortunate that his Wrst two PhD students, Professors Hans Coster and Peter Barry, both of whom are still active members of the society, presented lectures of appreciation in the session, along with Dr. Fred Chow who worked with Alex Hope for the last 20 years of his extraordinarily productive life. Alex Hope spent a considerable time working on the giant cells of the alga, Chara; and his work did much to popularise this species as a model system for electrophysiological studies of isolated cells. Chara is in active use today as evidenced by, for example, the article by Dr. Mary Beilby and colleagues. Alex Hope’s work on the biophysics of membrane transport inspired generations of biophysicists in Australia and beyond; indeed biophysical studies of membrane transport has remained a strength of the Australian Biophysics community as is reXected in many of the articles in this special edition. A traditionally informative feature of meetings of the Australian Society for Biophysics (and of course others) is the Poster Sessions: All poster presenters are invited to speak for 90 s in the poster hall, to introduce their work and entice closer scrutiny during the main poster sessions. This has been a very eVective way of generating interest in the posters and stimulating questions and debate on site. We are also very pleased that 5 of the articles in this issue come from students; Asi Cohen (student oral prize winner), Sophia Goodchild (Poster prize winner), David Szekely (Poster prize winner), HanShen Tae and Karunasekara Yamuna. We hope that you the reader will Wnd, in the following pages, amongst the fruit of the ASB garden, much that is of interest and relevance to your own study and work, in the area of membranes, membrane proteins and the structural and functional connections between these macromolecules and multimolecular assemblies. J. I. Vandenberg (&) Department of Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia e-mail: j.vandenberg@victorchang.edu.au