The inositol hexakisphosphate kinase family. Catalytic flexibility and function in yeast vacuole biogenesis

Saiardi et al. (Saiardi, A., Erdjument-Bromage, H., Snowman, A., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1326) previously described the cloning of a kinase from yeast and two kinases from mammals (types 1 and 2), which phosphorylate inositol hexakisphosphate (InsP(6)) to diphosphoinositol pentakisphosphate, a "high energy" candidate regulator of cellular trafficking. We have now studied the significance of InsP(6) kinase activity in Saccharomyces cerevisiae by disrupting the kinase gene. These ip6kDelta cells grew more slowly, their levels of diphosphoinositol polyphosphates were 60-80% lower than wild-type cells, and the cells contained abnormally small and fragmented vacuoles. Novel activities of the mammalian and yeast InsP(6) kinases were identified; inositol pentakisphosphate (InsP(5)) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP(4)), which was further metabolized to a novel compound, tentatively identified as bis-diphosphoinositol trisphosphate. The latter is a new substrate for human diphosphoinositol polyphosphate phosphohydrolase. Kinetic parameters for the mammalian type 1 kinase indicate that InsP(5) (K(m) = 1.2 micrometer) and InsP(6) (K(m) = 6.7 micrometer) compete for phosphorylation in vivo. This is the first time a PP-InsP(4) synthase has been identified. The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP(6). Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged.     

Superoxide dismutase C affects Dictyostelium contractile vacuole biogenesis and function

Dictyostelium cells cope with hypo-osmotic stress with a contractile vacuole (CV) system, which consists of one or two vacuoles that cyclically charge and discharge. Uniquely, a F-Actin remodeling dependent minimal mixing of the CV membrane components with the target plasmalemma during the fusion and the dischargement warrants the integrity of the CV bladder for an efficient next CV cycle. The effect of hypo-osmotic stress on F-Actin remodeling activity, however, is currently not well understood. Dictyostelium cells increase the level of intracellular superoxide level in response to hypo-osmotic stress, which in turn activates redox-sensitive Ras proteins, but not Akt, which is one of the Ras downstream targets and a major regulator of F-Actin remodeling. However, Akt is not insulated from the active Ras in cells lacking Superoxide dismutase C (SodC). We report here that sodC^- cells were compromised in the CV structure and function and the attenuation of Ras/PI3K/Akt signaling in several independent means significantly improved the compromised CV structure but not the function. Interestingly, when sodC^- cells were treated with 5-(N,N-Dimethyl) amiloride hydrochloride (EIPA), an inhibitor of sodium proton exchanger (NHE), both the structure and the function of the CV improved. Thus, a proper CV biogenesis in sodC^- cells was insufficient to restore their CV function, which in turn indicates the presence of an additional target for SodC and EIPA that modulates CV function.     

Taking possession: biogenesis of the Salmonella-containing vacuole

The Gram-negative pathogen Salmonella enterica can survive and replicate within a variety of mammalian cells. Regardless of the cell type, internalized bacteria survive and replicate within the Salmonella -containing vacuole, the biogenesis of which is dependent on bacterially encoded virulence factors. In particular, Type III secretion systems translocate bacterial effector proteins into the eukaryotic cell where they can specifically interact with a variety of targets. Salmonella has two distinct Type III secretion systems that are believed to have completely different functions. The SPI2 system is induced intracellularly and is required for intracellular survival in macrophages; it plays no role in invasion but is categorized as being required for Salmonella -containing vacuole biogenesis. In contrast, the SPI1 Type III secretion system is induced extracellularly and is essential for invasion of nonphagocytic cells. Its role in post-invasion processes has not been well studied. Recent studies indicate that Salmonella -containing vacuole biogenesis may be more dependent on SPI1 than previously believed. Other non-SPI2 virulence factors and the host cell itself may play critical roles in determining the intracellular environment of this facultative intracellular pathogen. In this review we discuss the recent advances in determining the mechanisms by which Salmonella regulate Salmonella -containing vacuole biogenesis and the implications of these findings.     

Geminating pollen has tubular vacuoles, displays highly dynamic vacuole biogenesis, and requires VACUOLESS1 for proper function

Vacuoles perform multiple functions in plants, and VCL1 (VACUOLESS1) is essential for biogenesis with loss of expression in the vcl1 mutant leading to lethality. Vacuole biogenesis plays a prominent role in gametophytes, yet is poorly understood. Given the importance of VCL1, we asked if it contributes to vacuole biogenesis during pollen germination. To address this question, it was essential to first understand the dynamics of vacuoles. A tonoplast marker, delta-TIP::GFP, under a pollen-specific promoter permitted the examination of vacuole morphology in germinating pollen of Arabidopsis. Our results demonstrate that germination involves a complex, yet definable, progression of vacuole biogenesis. Pollen vacuoles are extremely dynamic with remarkable features such as elongated (tubular) vacuoles and highly mobile cytoplasmic invaginations. Surprisingly, vcl1 did not adversely impact vacuole morphology in pollen germinated in vitro. To focus further on VCL1 in pollen, reciprocal backcrosses demonstrated reduced transmission of vcl1 through male gametophytes, indicating that vcl1 was expressive after germination. Interestingly, vcl1 affected the fertility of female gametophytes that undergo similarly complex vacuole biogenesis. Our results indicate that vcl1 is lethal in the sporophyte but is not fully expressive in the gametophytes. They also point to the complexity of pollen vacuoles and suggest that the mechanism of vacuole biogenesis in pollen may differ from that in other plant tissues.     

Plasmodesmata: structure, function and biogenesis

Plasmodesmata remain one of the outstanding mysteries in plant biology. In providing conduits for the exchange of small and large, informational molecules they are central to the growth, development and defence of all higher plants. In the past few years, strategies have been devised for the molecular dissection of plasmodesmal composition and function, and we are beginning to see how these enigmatic structures will become to be understood.     

The fungal cell wall: Modern concepts of its composition and biological function

This review deals with the cell wall (CW), a poorly known surface structure of the cell of mycelial fungi. Data are presented concerning (i) isolation techniques and purity control methods securing the absence of the cytoplasm content in the CW fraction and (ii) the chemical composition of the CW. The structural (backbone) and intrastructural components of the CW, such as aminopolysaccharides, α- and β-glucans, proteins, lipids, uronic acids, hydrophobins, sporopollenin, and melanins, are discussed in detail. Special attention is given to chitin and its novel function in terms of protecting the cells from stress as well as to the differences of this fungal aminopolysaccharide from the chitin of algae and Arthropoda. The apical growth of hyphae and the involvement of special microvesicles in morphogenesis of a fungal cell are discussed. Data on the enzymes involved in CW synthesis and lysis are presented. In conclusion, the functional role of the fungal CW is discussed in juxtaposition to the surface structures of higher eukaryotes.     

Small RNAs: classification, biogenesis, and function

Eukaryotes produce various types of small RNAs of 19-28 nt in length. With rapidly increasing numbers of small RNAs listed in recent years, we have come to realize how widespread their functions are and how diverse the biogenesis pathways have evolved. At the same time, we are beginning to grasp the common features and rules governing the key steps in small RNA pathways. In this review, I will summarize the current classification, biogenesis, action mechanism and function of these fascinating molecules.     

MicroRNAs: genomics, biogenesis, mechanism, and function

MicroRNAs (miRNAs) are endogenous approximately 22 nt RNAs that can play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.     

Vacuole biogenesis and protein transport to the plant vacuole: A comparison with the yeast vacuole and the mammalian lysosome

Summary The vacuole is often termed the lytic compartment of the plant cell. The yeast cell also possesses a vacuole containing acid hydrolases. In animal cells these enzymes are localized in the lysosome. Recent research suggests that there is good reason to regard these organelles as homologous in terms of protein transport. Although sorting motifs for the recognition of vacuolar proteins within the endomembrane system differ between the three organelles, there is an underlying similarity in targeting determinants in the cytoplasmic tails of Golgi-based receptors. In all three cases these determinants appear to interact with adaptins of clathrin-coated vesicles which ferry their cargo first of all to an endosomal compartment. The situation in sorting and targeting of plant vacuolar proteins is complicated by the fact that storage and lytic vacuoles may exist together in the same cell. The origin of these two types of vacuole is also a matter of some uncertanity.Abbrevations AP assembly protein - ALP alkaline phosphatase - ARF adenosine diphosphate ribosylation factor - BiP immunoglobulin binding protein - CCV clathrin coated vesicle - CPY carboxypeptidase-Y - DPAP dipeptidyl aminopeptidase - ER endoplasmic reticulum - GApp Golgi apparatus - LAMPs lysosomal associated membrane protein(s) - LAP lysosomal acid phosphatase - LIMPs lysosomal integral membrane protein(s) - MPRs mannosyl 6-phosphate receptors - MVB multivesicular bodies - NSF N-ethylmaleimide sensitive fusion (protein) - PAT phosphinotricine acetyltransferase - PB protein body - PHA phytohemagglutinin - PM plasma membrane - PSV protein storage vacuole - SNAPs soluble NSF attachment protein(s) - SNAREs SNAP receptor(s) - TGN trans Golgi network - TIP tonoplast integral protein - VPS vacuolar protein sorting - ZIO zinc iodide/osmium     

Compartments within a compartment: What C. elegans can tell us about ciliary subdomain composition,biogenesis, function,and disease

The primary cilium has emerged as a hotbed of sensory and developmental signaling, serving as a privileged domain to concentrate the functions of a wide number of channels, receptors and downstream signal transducers. This realization has provided important insight into the pathophysiological mechanisms underlying the ciliopathies, an ever expanding spectrum of multi-symptomatic disorders affecting the development and maintenance of multiple tissues and organs. One emerging research focus is the subcompartmentalised nature of the organelle, consisting of discrete structural and functional subdomains such as the periciliary membrane/basal body compartment, the transition zone, the Inv compartment and the distal segment/ciliary tip region. Numerous ciliopathy, transport-related and signaling molecules localize at these compartments, indicating specific roles at these subciliary sites. Here, by focusing predominantly on research from the genetically tractable nematode C. elegans, we review ciliary subcompartments in terms of their structure, function, composition, biogenesis and relationship to human disease.     

Microscopy and cytochemistry of the biogenesis of the parasitophorous vacuole

Some parasitic protozoa are able to penetrate into host cells where they multiply. The process of penetration involves steps such as attachment to the host cell surface, internalization of the protozoan through an endocytic process with the formation of a parasitophorous vacuole (PV), and the subsequent interaction of the protozoan with the membrane lining the PV. This review analyzes the biogenesis of the PV from a morphological and cytochemical perspective. Special emphasis is given to (a) the localization of plasma membrane-associated enzymes such as Na⁺-K⁺-ATPase, Ca²⁺-ATPase, 5-nucleotidase, and NAD(P)H-oxidase, (b) glycoconjugates, detected using labeled lectins, (c) anionic sites, detected using cationic particles, and (d) integral membrane proteins, using freeze-fracture replicas, and lipids during the formation of the PV containing Trypanosoma cruzi, Leishmania, Toxoplasma gondii, and Plasmodium.     

Extracellular Streptomyces lividans vesicles: composition,biogenesis and antimicrobial activity

We selected Streptomyces lividans to elucidate firstly the biogenesis and antimicrobial activities of extracellular vesicles that a filamentous and highly differentiated Gram‐positive bacterium produces. Vesicle types range in diameter from 110 to 230 nm and 20 to 60 nm, respectively; they assemble to clusters, and contain lipids and phospholipids allowing their in situ imaging by specific fluorescent dyes. The presence of the identified secondary metabolite undecylprodigiosin provokes red fluorescence of a portion of the heterogeneous vesicle populations facilitating in vivo monitoring. Protuberances containing vesicles generate at tips, and alongside of substrate hyphae, and enumerate during late vegetative growth to droplet‐like exudates. Owing to in situ imaging in the presence and absence of a green fluorescent vancomycin derivative, we conclude that protuberances comprising vesicles arise at sites with enhanced levels of peptidoglycan subunits [pentapeptide of lipid II (C55)‐linked disaccharides], and reduced levels of polymerized and cross‐linked peptidoglycan within hyphae. These sites correlate with enhanced levels of anionic phospholipids and lipids. Vesicles provoke pronounced damages of Aspergillus proliferans, Verticillium dahliae and induced clumping and distortion of Escherichia coli. These harmful effects are likely attributable to the action of the identified vesicular compounds including different enzyme types, components of signal transduction cascades and undecylprodigiosin. Based on our pioneering findings, we highlight novel clues with environmental implications and application potential.     

Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis.

vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.     

Plant peroxisomes: biogenesis and function

Peroxisomes are eukaryotic organelles that are highly dynamic both in morphology and metabolism. Plant peroxisomes are involved in numerous processes, including primary and secondary metabolism, development, and responses to abiotic and biotic stresses. Considerable progress has been made in the identification of factors involved in peroxisomal biogenesis, revealing mechanisms that are both shared with and diverged from non-plant systems. Furthermore, recent advances have begun to reveal an unexpectedly large plant peroxisomal proteome and have increased our understanding of metabolic pathways in peroxisomes. Coordination of the biosynthesis, import, biochemical activity, and degradation of peroxisomal proteins allows for highly dynamic responses of peroxisomal metabolism to meet the needs of a plant. Knowledge gained from plant peroxisomal research will be instrumental to fully understanding the organelle's dynamic behavior and defining peroxisomal metabolic networks, thus allowing the development of molecular strategies for rational engineering of plant metabolism, biomass production, stress tolerance, and pathogen defense.     

Spliceosomal UsnRNP biogenesis, structure and function

Significant advances have been made in elucidating the biogenesis pathway and three-dimensional structure of the UsnRNPs, the building blocks of the spliceosome. U2 and U4/U6*U5 tri-snRNPs functionally associate with the pre-mRNA at an earlier stage of spliceosome assembly than previously thought, and additional evidence supporting UsnRNA-mediated catalysis of pre-mRNA splicing has been presented.