A Retention Signal Necessary and Sufficient for Endoplasmic Reticulum Localization Maps to the Transmembrane Domain of Hepatitis C Virus Glycoprotein E2

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which is retained in the endoplasmic reticulum (ER). To identify whether E1 and/or E2 contains an ER-targeting signal potentially involved in ER retention of the E1-E2 complex, these proteins were expressed alone and their intracellular localization was studied. Due to misfolding of E1 in the absence of E2, no conclusion on the localization of its native form could be drawn from the expression of E1 alone. E2 expressed in the absence of E1 was shown to be retained in the ER similarly to E1-E2 complex. Chimeric proteins in which E2 domains were exchanged with corresponding domains of a protein normally transported to the plasma membrane (CD4) were constructed to identify the sequence responsible for its ER retention. The transmembrane domain (TMD) of E2 (C-terminal 29 amino acids) was shown to be sufficient for retention of the ectodomain of CD4 in the ER compartment. Replacement of the E2 TMD by the anchor signal of CD4 or a glycosyl phosphatidylinositol (GPI) moiety led to its expression on the cell surface. In addition, replacement of the E2 TMD by the anchor signal of CD4 or a GPI moiety abolished the formation of E1-E2 complexes. Together, these results suggest that, besides having a role as a membrane anchor, the TMD of E2 is involved in both complex formation and intracellular localization.Hepatitis C virus (HCV) is an enveloped virus which belongs to the Flaviviridae family (15). Its genome encodes two membrane-associated envelope glycoproteins (E1 and E2). E1 and E2 glycoproteins interact to form a complex which has been proposed as a functional subunit of HCV virions (11, 17, 26, 41). Characterization of HCV glycoprotein complex formation expressed by using the vaccinia/T7 or Sindbis virus system indicates that a majority of HCV glycoproteins are misfolded (9, 11). Recently, we have produced a monoclonal antibody (MAb) which recognizes properly folded E2 and precipitates native HCV glycoprotein complexes but not misfolded aggregates (9). Properly folded E1 and E2 interact to form a heterodimer stabilized by noncovalent interactions, and the kinetics of association between E1 and E2 indicate that the formation of stable E1-E2 complexes is slow (half-time of association [t_1/2] ≈ 2 h). The folding of E1 and E2 has been studied and indicates that formation of intramolecular disulfide bonds is slow for E1 (t_1/2 > 1 h) whereas it is rapid for E2 (12). By using human and mouse MAbs, it has been shown that folding of a subdomain(s) of E2 correlates with acquisition of intramolecular disulfide bonds but that complete folding of E2 is slow (t_1/2 ≈ 2 h) (9, 19). In addition, E1 expressed in the absence of E2 does not fold properly, suggesting that E2 plays a chaperone-like role in the folding of E1 (32).The HCV glycoproteins are heavily modified by N-linked glycosylation and contain hydrophobic domains in their carboxy-terminal regions acting presumably as membrane anchors, giving the proteins a type I membrane topology (43). The E2 glycoprotein extends to residue 746 (position on the polyprotein), and deletions of at least 31 C-terminal amino acids lead to its secretion (47). This is in accordance with other data proposing that the hydrophobic anchor domain begins at amino acid 718 (33). However, only a shorter secreted form of E2 glycoprotein ending at amino acid 661 appears to be properly folded (32). For E1, a larger deletion (71 amino acids) seems to be necessary for its secretion, but this secreted protein is not properly folded (32).Due to the lack of an efficient cell culture replication system, HCV particle assembly and release have not been examined directly. However, the lack of complex glycans, the endoplasmic reticulum (ER) localization of expressed HCV glycoproteins (11, 41), and the absence of these proteins on the cell surface (11, 49) suggest that initial virion morphogenesis may occur by budding into intracellular vesicles. More recently, we have confirmed that the mature E1-E2 heterodimer does not leave the ER, suggesting that E1 and/or E2 contains a signal for retention of the heterodimer in this compartment (9).In this study, we show that E2 glycoprotein expressed alone is retained in the ER similarly to the E1-E2 heterodimer and that a signal for ER retention of E2 resides in its transmembrane domain (TMD) (C-terminal 29 amino acids). The evidence for this retention signal was derived from expression of chimeric proteins in which E2 domains were exchanged with corresponding domains of a protein normally transported to the plasma membrane (CD4).     

Yeast Phosphofructokinase-1 Subunit Pfk2p Is Necessary for pH Homeostasis and Glucose-dependent Vacuolar ATPase Reassembly

V-ATPases are conserved ATP-driven proton pumps that acidify organelles. Yeast V-ATPase assembly and activity are glucose-dependent. Glucose depletion causes V-ATPase disassembly and its inactivation. Glucose readdition triggers reassembly and resumes proton transport and organelle acidification. We investigated the roles of the yeast phosphofructokinase-1 subunits Pfk1p and Pfk2p for V-ATPase function. The pfk1Δ and pfk2Δ mutants grew on glucose and assembled wild-type levels of V-ATPase pumps at the membrane. Both phosphofructokinase-1 subunits co-immunoprecipitated with V-ATPase in wild-type cells; upon deletion of one subunit, the other subunit retained binding to V-ATPase. The pfk2Δ cells exhibited a partial vma growth phenotype. In vitro ATP hydrolysis and proton transport were reduced by 35% in pfk2Δ membrane fractions; they were normal in pfk1Δ. In vivo, the pfk1Δ and pfk2Δ vacuoles were alkalinized and the cytosol acidified, suggestive of impaired V-ATPase proton transport. Overall the pH alterations were more dramatic in pfk2Δ than pfk1Δ at steady state and after readdition of glucose to glucose-deprived cells. Glucose-dependent reassembly was 50% reduced in pfk2Δ, and the vacuolar lumen was not acidified after reassembly. RAVE-assisted glucose-dependent reassembly and/or glucose signals were disturbed in pfk2Δ. Binding of disassembled V-ATPase (V₁ domain) to its assembly factor RAVE (subunit Rav1p) was 5-fold enhanced, indicating that Pfk2p is necessary for V-ATPase regulation by glucose. Because Pfk1p and Pfk2p are necessary for V-ATPase proton transport at the vacuole in vivo, a role for glycolysis at regulating V-ATPase proton transport is discussed.     

A Conserved Functional Domain of Drosophila Coracle Is Required for Localization at the Septate Junction and Has Membrane-organizing Activity

The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members.     

Sla2p Is Associated with the Yeast Cortical Actin Cytoskeleton via Redundant Localization Signals

Sla2p, also known as End4p and Mop2p, is the founding member of a widely conserved family of actin-binding proteins, a distinguishing feature of which is a C-terminal region homologous to the C terminus of talin. These proteins may function in actin cytoskeleton-mediated plasma membrane remodeling. A human homologue of Sla2p binds to huntingtin, the protein whose mutation results in Huntington's disease. Here we establish by immunolocalization that Sla2p is a component of the yeast cortical actin cytoskeleton. Deletion analysis showed that Sla2p contains two separable regions, which can mediate association with the cortical actin cytoskeleton, and which can provide Sla2p function. One localization signal is actin based, whereas the other signal is independent of filamentous actin. Biochemical analysis showed that Sla2p exists as a dimer in vivo. Two-hybrid analysis revealed two intramolecular interactions mediated by coiled-coil domains. One of these interactions appears to underlie dimer formation. The other appears to contribute to the regulation of Sla2p distribution between the cytoplasm and plasma membrane. The data presented are used to develop a model for Sla2p regulation and interactions.     

Requirement of Sequences outside the Conserved Kinase Domain of Fission Yeast Rad3p for Checkpoint Control

The fission yeast Rad3p checkpoint protein is a member of the phosphatidylinositol 3-kinase-related family of protein kinases, which includes human ATMp. Mutation of the ATM gene is responsible for the disease ataxia-telangiectasia. The kinase domain of Rad3p has previously been shown to be essential for function. Here, we show that although this domain is necessary, it is not sufficient, because the isolated kinase domain does not have kinase activity in vitro and cannot complement a rad3 deletion strain. Using dominant negative alleles of rad3, we have identified two sites N-terminal to the conserved kinase domain that are essential for Rad3p function. One of these sites is the putative leucine zipper, which is conserved in other phosphatidylinositol 3-kinase-related family members. The other is a novel motif, which may also mediate Rad3p protein-protein interactions.     

A Conserved Rubredoxin Is Necessary for Photosystem II Accumulation in Diverse Oxygenic Photoautotrophs

In oxygenic photosynthesis, two photosystems work in tandem to harvest light energy and generate NADPH and ATP. Photosystem II (PSII), the protein-pigment complex that uses light energy to catalyze the splitting of water, is assembled from its component parts in a tightly regulated process that requires a number of assembly factors. The 2pac mutant of the unicellular green alga Chlamydomonas reinhardtii was isolated and found to have no detectable PSII activity, whereas other components of the photosynthetic electron transport chain, including photosystem I, were still functional. PSII activity was fully restored by complementation with the RBD1 gene, which encodes a small iron-sulfur protein known as a rubredoxin. Phylogenetic evidence supports the hypothesis that this rubredoxin and its orthologs are unique to oxygenic phototrophs and distinct from rubredoxins in Archaea and bacteria (excluding cyanobacteria). Knockouts of the rubredoxin orthologs in the cyanobacterium Synechocystis sp. PCC 6803 and the plant Arabidopsis thaliana were also found to be specifically affected in PSII accumulation. Taken together, our data suggest that this rubredoxin is necessary for normal PSII activity in a diverse set of organisms that perform oxygenic photosynthesis.     

A Conserved Domain in the Coronavirus Membrane Protein Tail Is Important for Virus Assembly

Coronavirus membrane (M) proteins play key roles in virus assembly, through M-M, M-spike (S), and M-nucleocapsid (N) protein interactions. The M carboxy-terminal endodomain contains a conserved domain (CD) following the third transmembrane (TM) domain. The importance of the CD (SWWSFNPETNNL) in mouse hepatitis virus was investigated with a panel of mutant proteins, using genetic analysis and transient-expression assays. A charge reversal for negatively charged E₁₂₁ was not tolerated. Lysine (K) and arginine (R) substitutions were replaced in recovered viruses by neutrally charged glutamine (Q) and leucine (L), respectively, after only one passage. E₁₂₁Q and E₁₂₁L M proteins were capable of forming virus-like particles (VLPs) when coexpressed with E, whereas E₁₂₁R and E₁₂₁K proteins were not. Alanine substitutions for the first four or the last four residues resulted in viruses with significantly crippled phenotypes and proteins that failed to assemble VLPs or to be rescued into the envelope. All recovered viruses with alanine substitutions in place of SWWS residues had second-site, partially compensating, changes in the first TM of M. Alanine substitution for proline had little impact on the virus. N protein coexpression with some M mutants increased VLP production. The results overall suggest that the CD is important for formation of the viral envelope by helping mediate fundamental M-M interactions and that the presence of the N protein may help stabilize M complexes during virus assembly.Coronaviruses are widespread, medically important respiratory and enteric pathogens of humans and a wide range of animals. New human coronaviruses (HCoV), including severe acute respiratory syndrome CoV (SARS-CoV), HCoV-NL63, and HCoV-HKU1, were recently identified (40, 47). The potential for emergence of other new viruses and the zoonotic nature of some coronaviruses strongly warrants understanding old and new viruses. Understanding vital interactions that take place during virus assembly and conserved domains (CDs) that mediate these interactions can provide insight toward identification of targets for development of antiviral therapeutics and vaccines.Coronaviruses are enveloped positive-stranded RNA viruses that belong to the Coronaviridae family in the Nidovirales order. The virion envelope contains at least three structural proteins, the membrane (M), spike (S), and envelope (E) proteins. The genomic RNA is encapsidated by the N phosphoprotein to form a helical nucleocapsid. The S glycoprotein is the viral attachment protein that facilitates infection through fusion of viral and cellular membranes and is the major target of neutralizing antibodies (13). The M glycoprotein is the most abundant component of the viral envelope and plays required, key roles in virus assembly (9, 20, 31, 33, 41). The E protein is a minor component of the viral envelope that plays an important, not clearly defined, role(s) during virus assembly and release (2, 5, 41).Coronavirus M proteins are divergent in their amino acid content, but all share the same overall basic structural characteristics. The proteins have three TM domains, flanked by a short amino-terminal glycosylated domain and a long carboxy-terminal tail located outside and inside the virion, respectively (14) (Fig. ​(Fig.11 A). M localizes in the Golgi region when expressed alone (20, 22). M molecules interact with each other and also with the spike and nucleocapsid during virus assembly (8-10, 23, 31, 33). M-M interactions constitute the overall scaffold for the viral envelope. The S protein and a small number of E molecules are interspersed in the M protein lattice in mature virions. Previous studies from a number of labs implicated multiple M domains and residues as being important for coronavirus assembly (6, 8, 9, 17, 43). Coronaviruses assemble and bud at intracellular membranes in the region of the endoplasmic reticulum (ER) Golgi intermediate compartment (ERGIC) (22, 39). Coexpression of only the M and the E proteins is sufficient for virus-like particle (VLP) assembly for most coronaviruses (2, 41).Open in a separate windowFIG. 1.M protein conserved domain and mutants. (A) A linear schematic of the M protein illustrating the relative positions of the three TM domains (black boxes) and the position of the CD in the tail. (B) Alignment of CDs from representative coronaviruses. Full-length amino acid sequences from transmissible gastroenteritis virus (TGEV), feline coronavirus (FeCoV), human coronavirus 229E, human coronavirus NL63, mouse hepatitis virus (MHV), bovine coronavirus (BCoV), human coronavirus OC43, porcine hemagglutinating encephalomyelitis virus (HEV), human coronavirus HKU1, SARS-CoV, infectious bronchitis virus (IBV), and turkey coronavirus (TCoV) were aligned by using CLUSTAL W (25). (C) Mutations introduced into the MHV CD, with + and − symbols used to indicate VLP production and virus recovery for each mutant.The long intravirion (cytoplasmic) tail of M consists of an amphipathic domain following the third TM and a short hydrophilic region at the carboxyl end of the tail (Fig. ​(Fig.1A).1A). The amphipathic domain appears to be closely associated with the membrane (34). At the amino terminus of the amphipathic domain, there is a highly conserved 12-amino-acid domain (SWWSFNPETNNL), consisting of residues 114 to 125 in the mouse hepatitis virus (MHV) A59 M protein (Fig. ​(Fig.1B)1B) (19). These residues are almost identically conserved across the entire Coronaviridae family. Because of the crucial role that M plays in virus assembly and the high conservation of this domain, we hypothesized that it is functionally important for virus assembly. To test this, a series of changes were introduced in the CD. The functional impact of the changes was studied in the context of the virus by genetic analysis and the ability of the mutant M proteins to participate in VLP assembly. The results show that the CD is functionally important for M protein to participate in virus assembly. The domain may help mediate important lateral interactions between M molecules. The results suggest that the N protein helps stabilize M complexes during virus assembly.     

Mdm12p, a Component Required for Mitochondrial Inheritance That Is Conserved between Budding and Fission Yeast

Saccharomyces cerevisiae cells lacking the MDM12 gene product display temperature-sensitive growth and possess abnormally large, round mitochondria that are defective for inheritance by daughter buds. Analysis of the wild-type MDM12 gene revealed its product to be a 31-kD polypeptide that is homologous to a protein of the fission yeast Schizosaccharomyces pombe. When expressed in S. cerevisiae, the S. pombe Mdm12p homolog conferred a dominant-negative phenotype of giant mitochondria and aberrant mitochondrial distribution, suggesting partial functional conservation of Mdm12p activity between budding and fission yeast. The S. cerevisiae Mdm12p was localized by indirect immunofluorescence microscopy and by subcellular fractionation and immunodetection to the mitochondrial outer membrane and displayed biochemical properties of an integral membrane protein. Mdm12p is the third mitochondrial outer membrane protein required for normal mitochondrial morphology and distribution to be identified in S. cerevisiae and the first such mitochondrial component that is conserved between two different species.     

Spa2p Interacts with Cell Polarity Proteins and Signaling Components Involved in Yeast Cell Morphogenesis

The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth. To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2Δ, pea2Δ, and bud6Δ mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes. Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton. Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites. One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region contains a conserved domain, SHD-II. Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defects, suggesting that other regions are required for Spa2p function. By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth. Spa2p interacts with Ste11p (MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was mapped to the N-terminal putative regulatory domain. Bud6p interacts with Ste11p. The MEK-interacting region of Spa2p corresponds to the highly conserved SHD-I domain, which is shown to be important for mating and MAPK signaling. spa2 mutants exhibit reduced levels of pheromone signaling and an elevated level of Slt2p kinase activity. We thus propose that Spa2p, Pea2p, and Bud6p function together, perhaps as a complex, to promote polarized morphogenesis through regulation of the actin cytoskeleton and signaling pathways.     

A Conserved Domain Is Crucial for Acceptor Substrate Binding in a Family of Glucosyltransferases

Serine-rich repeat glycoproteins (SRRPs) are highly conserved in streptococci and staphylococci. Glycosylation of SRRPs is important for bacterial adhesion and pathogenesis. Streptococcus agalactiae is the leading cause of bacterial sepsis and meningitis among newborns. Srr2, an SRRP from S. agalactiae strain COH1, has been implicated in bacterial virulence. Four genes (gtfA, gtfB, gtfC, and gtfD) located downstream of srr2 share significant homology with genes involved in glycosylation of other SRRPs. We have shown previously that gtfA and gtfB encode two glycosyltransferases, GtfA and GtfB, that catalyze the transfer of GlcNAc residues to the Srr2 polypeptide. However, the function of other glycosyltransferases in glycosylation of Srr2 is unknown. In this study, we determined that GtfC catalyzed the direct transfer of glucosyl residues to Srr2-GlcNAc. The GtfC crystal structure was solved at 2.7 Å by molecular replacement. Structural analysis revealed a loop region at the N terminus as a putative acceptor substrate binding domain. Deletion of this domain rendered GtfC unable to bind to its substrate Srr2-GlcNAc, concurrently abolished the glycosyltransferase activity of GtfC, and also altered glycosylation of Srr2. Furthermore, deletion of the corresponding regions from GtfC homologs also abolished their substrate binding and enzymatic activity, indicating that this region is functionally conserved. In summary, we have determined that GtfC is important for the glycosylation of Srr2 and identified a conserved loop region that is crucial for acceptor substrate binding from GtfC homologs in streptococci. These findings shed new mechanistic insight into this family of glycosyltransferases.     

A Conserved Interaction between the SDI Domain of Bre2 and the Dpy-30 Domain of Sdc1 Is Required for Histone Methylation and Gene Expression

In Saccharomyces cerevisiae, lysine 4 on histone H3 (H3K4) is methylated by the Set1 complex (Set1C or COMPASS). Besides the catalytic Set1 subunit, several proteins that form the Set1C (Swd1, Swd2, Swd3, Spp1, Bre2, and Sdc1) are also needed to mediate proper H3K4 methylation. Until this study, it has been unclear how individual Set1C members interact and how this interaction may impact histone methylation and gene expression. In this study, Bre2 and Sdc1 are shown to directly interact, and it is shown that the association of this heteromeric complex is needed for proper H3K4 methylation and gene expression to occur. Interestingly, mutational and biochemical analysis identified the C terminus of Bre2 as a critical protein-protein interaction domain that binds to the Dpy-30 domain of Sdc1. Using the human homologs of Bre2 and Sdc1, ASH2L and DPY-30, respectively, we demonstrate that the C terminus of ASH2L also interacts with the Dpy-30 domain of DPY-30, suggesting that this protein-protein interaction is maintained from yeast to humans. Because of the functionally conserved nature of the C terminus of Bre2 and ASH2L, this region was named the SDI (Sdc1 Dpy-30 interaction) domain. Finally, we show that the SDI-Dpy-30 domain interaction is physiologically important for the function of Set1 in vivo, because specific disruption of this interaction prevents Bre2 and Sdc1 association with Set1, resulting in H3K4 methylation defects and decreases in gene expression. Overall, these and other mechanistic studies on how H3K4 methyltransferase complexes function will likely provide insights into how human MLL and SET1-like complexes or overexpression of ASH2L leads to oncogenesis.     

A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase

Phosphatidylinositol phosphate kinase (PIPK) is an enzyme involved in the regulation of cellular levels of phosphoinositides involved in various physiological processes, such as cytoskeletal organization, ion channel activation, and vesicle trafficking. In animals, research has focused on the modes of activation and function of PIPKs, providing an understanding of the importance of plasma membrane localization. However, it still remains unclear how this issue is regulated in plant PIPKs. Here, we demonstrate that the carboxyl-terminal catalytic domain, which contains the activation loop, is sufficient for plasma membrane localization of PpPIPK1, a type I/II B PIPK from the moss Physcomitrella patens. The importance of the carboxyl-terminal catalytic domain for plasma membrane localization was confirmed with Arabidopsis (Arabidopsis thaliana) AtPIP5K1. Our findings, in which substitution of a conserved dibasic amino acid pair in the activation loop of PpPIPK1 completely prevented plasma membrane targeting and abolished enzymatic activity, demonstrate its critical role in these processes. Placing our results in the context of studies of eukaryotic PIPKs led us to conclude that the function of the dibasic amino acid pair in the activation loop in type I/II PIPKs is plant specific.Phosphoinositides (PIs) are minor lipids found in membrane fractions but implicated in a wide variety of physiological regulations in eukaryotes (Di Paolo and De Camilli, 2006; Zonia and Munnik, 2006). Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] is a major PI in animal plasma membranes, affecting the localization and activity of various kinds of proteins carrying phosphatidylinositol-binding domains, which in turn affect the regulation of cytoskeletal organization, vesicle trafficking, cell proliferation, and cell growth during development and stress responses (Doughman et al., 2003; Downes et al., 2005; Di Paolo and De Camilli, 2006; Zonia and Munnik, 2006; Heck et al., 2007). In addition, PtdIns(4,5)P₂ is also a well-known substrate of phospholipase C, producing second messengers such as diacylglycerol, phosphatidic acid (PA), and inositol-1,4,5-trisphosphate, which are involved in the activation of intracellular signal transduction pathways (Zonia and Munnik, 2006). Transient accumulation of PtdIns(4,5)P₂ has also been observed under various kinds of environmental stress (Pical et al., 1999; DeWald et al., 2001), suggesting an important role of this lipid in the regulation of stress signal transduction pathways also in plants. These findings indicate that PtdIns(4,5)P₂ is multifunctional and involved in a variety of cellular processes. Therefore, elucidation of the mechanisms controlling the cellular levels of PtdIns(4,5)P₂ is important in understanding the significance of PI signaling in eukaryotes.PtdIns(4,5)P₂ is synthesized by phosphatidylinositol phosphate kinases (PIPKs; Anderson et al., 1999; Doughman et al., 2003; Heck et al., 2007). Physiological roles of several plant PIPKs have been reported. In Arabidopsis (Arabidopsis thaliana), AtPIP5K3 is an essential regulator of tip growth of root hairs (Kusano et al., 2008; Stenzel et al., 2008), while AtPIPK4 and AtPIPK5 are essential for pollen germination and pollen tube elongation (Ischebeck et al., 2008; Sousa et al., 2008). In addition, AtPIP5K9 was shown to interact with the cytosolic invertase CINV1 to regulate sugar-mediated root cell elongation negatively (Lou et al., 2007). Rice (Oryza sativa) OsPIPK1 is proposed to be involved in shoot growth and floral initiation through the regulation of floral induction genes (Ma et al., 2004). In animals, membrane-associated type I PIPK mainly phosphorylates the D-5 hydroxyl group of PtdIns4P to produce PtdIns(4,5)P₂ but also produces PtdIns(3,4)P₂ and PtdIns(3,5)P₂ from PtdIns3P with 5- and 4-kinase activity (Anderson et al., 1999; Heck et al., 2007), whereas type II PIPK prefers the D-4 position of PtdIns5P, producing PtdIns(4,5)P₂ in the nucleus and at the endoplasmic reticulum (Clarke et al., 2007). Thus, in animals, type I and II PIPKs are involved in the generation of PtdIns(4,5)P₂ via different pathways. Molecular biological analysis of plant PIPKs was initiated with AtPIP5K1 from Arabidopsis (Mikami et al., 1998), which phosphorylates PtdIns3P, PtdIns4P, and PtdIns(4,5)P₂ to produce PtdIns(3,4)P₂, PtdIns(4,5)P₂, and PtdIns (3,4,5)P₃, respectively, with D-4- and D-5-kinase activity (Elge et al., 2001; Westergren et al., 2001; Im et al., 2007). Similar enzymatic activity was also reported for other PIPKs from Arabidopsis (Ischebeck et al., 2008; Kusano et al., 2008; Stenzel et al., 2008). In addition, a PIPK from the moss Physcomitrella patens (designated as PpPIPK1) preferred PtdIns4P, PtdIns3P, and PtdIns(3,4)P₂ as substrates, but not PtdIns5P, producing PtdIns(4,5)P₂, PtdIns(3,4)P₂, and PtdIns(3,4,5)P₃, respectively (Saavedra et al., 2009). These findings indicate that the substrate specificity of plant PIPKs is essentially the same as that of type I PIPKs. However, AtPIP5K1 has yet to be classified as either type I or type II based on sequence comparisons of the catalytic domain (CD; Mikami et al., 1998). This was confirmed by a genome-wide analysis of PIPK genes in Arabidopsis in which all 11 PIPKs were classified as type I/II based on sequence comparisons of the CDs, which were further subdivided into subtypes A and B (Mueller-Roeber and Pical, 2002). Therefore, it is suggested that typical type I and II PIPKs are absent in plants, although further confirmation is needed.The conserved PIPK CD contains a short highly conserved region near its C-terminal end, designated the activation loop, which acts as the substrate-binding site and is responsible for the differences in substrate specificity and subcellular localization between animal type I and type II PIPKs (Kunz et al., 2000, 2002). Substrate specificities of animal type I and type II PIPKs, for example, are determined by a respective Glu and Ala at the corresponding positions in the activation loop. Moreover, it has been established that substitution of Glu to Ala results in a swap of substrate specificity and subcellular localization between the two types (Kunz et al., 2000, 2002). In contrast to animal PIPKs, a substitution in the activation loop of PpPIPK1 from Glu to Ala resulted in a nearly complete loss of type I/II activity; however, such a mutation did not fully convert the substrate specificity, although an enhancement of type II versus type I activity was observed (Saavedra et al., 2009). Since the corresponding amino acid residue is Glu in all plant PIPKs so far reported, it is suggested that there also is a plant-specific mode of substrate specificity regulation in plant type I/II PIPKs. However, enzymatic activity appears to be modified in similar ways between plant type I/II and animal type I PIPKs; that is, phosphorylation- and PA-dependent activation of PIPKs has been observed in both animals and plants (Moritz et al., 1992; Jenkins et al., 1994; Pical et al., 1999; Westergren et al., 2001; Perera et al., 2005; Saavedra et al., 2009).The regulation of plasma membrane localization of mammalian type I PIPKs remains confusing. In addition to the involvement of a Glu residue as mentioned above, the substitution of two Lys residues in the activation loop to Asn residues changes the subcellular localization from the plasma membrane to the cytosol (Kunz et al., 2000, 2002). However, Arioka et al. (2004) also showed that the plasma membrane localization of type I PIPKs is regulated by another basic amino acid pair localized downstream of the activation loop in the CD, which is not found in type II PIPKs. Interestingly, the mechanism behind plasma membrane localization of plant PIPKs seems to differ significantly from the animal one. The obvious structural feature of plant PIPKs is the presence of a repetition of membrane occupation and recognition nexus (MORN) motifs at the N-terminal half, which is conserved across the B subfamily of plant type I/II PIPKs (Mueller-Roeber and Pical, 2002). The MORN motif was first identified in mammalian junctophilin, an endoplasmic reticulum-membrane-bound component of the junctional complex between the plasma membrane and the endoplasmic reticulum (Takeshima et al., 2000). Since MORN motifs are not found in PIPKs from nonplant organisms, a plant-specific mode of PIPK activation is speculated. Indeed, a regulatory role of the MORN domain was reported in the enzymatic activation of AtPIP5K1 (Im et al., 2007) and in root hair formation, but not in enzymatic activation, of AtPIP5K3 (Stenzel et al., 2008). Moreover, the MORN domain may play a role in the plasma membrane localization of OsPIPK1 from rice and AtPIP5K1 and AtPIP5K3 from Arabidopsis (Ma et al., 2006; Im et al., 2007; Kusano et al., 2008). However, stable transformation of tobacco (Nicotiana tabacum) cells to express an AtPIP5K1 MORN domain-GFP fusion did not allow visualization of the plasma membrane localization of this protein (Im et al., 2007). Thus, it is not clear if the MORN domain functions as a plasma membrane-targeting module.Given the sequence conservation of the CD among eukaryotic PIPKs (Saavedra et al., 2009), we hypothesize that the CD is responsible for the plasma membrane localization of plant PIPKs. Thus, to gain further insight into the mechanisms regulating this issue, we dissected PpPIPK1 to determine the molecular determinants of plasma membrane localization. Here, we show that the MORN domain is not involved in the plasma membrane localization of PpPIPK1 and AtPIP5K1 in P. patens protoplasts and onion (Allium cepa) epidermal cells. We further demonstrate that two basic amino acids, but not Glu, conserved in the activation loop of the CD are required for plasma membrane localization. These findings demonstrate that the activation mode of type I/II PIPKs is plant specific and differs from that of the membrane-localized animal type I PIPKs.