The lipid kinase PI4KIIIß preserves lysosomal identity.

Lipid modifications are essential in cellular sorting and trafficking inside cells. The role of phosphoinositides in trafficking between Golgi and endocytic/lysosomal compartments has been extensively explored and the kinases responsible for these lipid changes have been identified. In contrast, the mechanisms that mediate exit and recycling from lysosomes (Lys), considered for a long time as terminal compartments, are less understood. In this work, we identify a dynamic association of the lipid kinase PI4KIIIß with Lys and unveil its regulatory function in lysosomal export and retrieval. We have found that absence of PI4KIIIß leads to abnormal formation of tubular structures from the lysosomal surface and loss of lysosomal constituents through these tubules. We demonstrate that the kinase activity of PI4KIIIß is necessary to prevent this unwanted lysosomal efflux under normal conditions, and to facilitate proper sorting when recycling of lysosomal material is needed, such as in the physiological context of lysosomal reformation after prolonged starvation.

Clathrin is required for postmitotic Golgi reassembly.

During the G2-M transition, the highly organized Golgi apparatus undergoes reversible fragmentation through unstacking of the cisternal ribbon and disassembly into radially dispersed vesicles and tubules. These Golgi-derived fragments redistribute randomly within the cytoplasm, partition stochastically, and in telophase coalesce to generate a functionally and structurally intact Golgi complex. Here we identified a novel step in postmitotic Golgi reassembly that requires the clathrin heavy chain (CHC). We used siRNA-mediated CHC knockdown, biochemistry, and morphological analysis and showed that the spindle- and spindle pole-associated clathrin pools are membrane-bound and required for postmitotic Golgi reassembly. The results presented here show that clathrin remains associated with the spindle poles throughout mitosis and that this clathrin pool is distinct from the previously characterized spindle-associated population. We suggest that clathrin may provide a template for postmitotic Golgi reassembly and cisternal remodeling. In absence of the CHC, the Golgi apparatus remained disconnected and disordered and failed to regain its characteristic perinuclear, lace-like morphology. Our findings build on previous independent reports that clathrin is required for Golgi reassembly following disruption with pharmacological agents and for mitotic chromosome congression.

The Golgi protein p115 associates with gamma-tubulin and plays a role in Golgi structure and mitosis progression.

The Golgi apparatus is a network of polarized cisternae localized to the perinuclear region in mammalian cells. It undergoes extensive vesiculation at the onset of mitosis and its reassembly requires factors that are in part segregated via the mitotic spindle. Here we show that unlike typical Golgi markers, the Golgi-protein p115 partitioned with the spindle poles throughout mitosis. An armadillo-fold in its N terminus mediated a novel interaction between p115 and γ-tubulin and functioned in its centrosomal targeting. Both the N- and C-terminal regions of p115 were required to maintain Golgi structure. Strikingly, p115 was essential for mitotic spindle function and the resolution of the cytokinetic bridge because its depletion resulted in spindle collapse, chromosome missegregation, and failed cytokinesis. We demonstrate that p115 plays a critical role in mitosis progression, implicating it as the only known golgin to regulate both mitosis and apoptosis.

Opposing action of casein kinase 1 and calcineurin in nucleo-cytoplasmic shuttling of mammalian translation initiation factor eIF6.

Eukaryotic initiation factor 6 (eIF6), a highly conserved protein from yeast to mammals, is essential for 60 S ribosome biogenesis and assembly. Both yeast and mammalian eIF6 are phosphorylated at Ser-174 and Ser-175 by the nuclear isoform of casein kinase 1 (CK1). The molecular basis of eIF6 phosphorylation, however, remains elusive. In the present work, we show that subcellular distribution of eIF6 in the nuclei and the cytoplasm of mammalian cells is mediated by dephosphorylation and phosphorylation, respectively. This nucleo-cytoplasmic shuttling is dependent on the phosphorylation status at Ser-174 and Ser-175 of eIF6. We demonstrate that Ca(2+)-activated calcineurin phosphatase binds to and promotes nuclear localization of eIF6. Increase in intracellular concentration of Ca(2+) leads to rapid translocation of eIF6 from the cytoplasm to the nucleus, an event that is blocked by specific calcineurin inhibitors cyclosporin A or FK520. Nuclear export of eIF6 is regulated by phosphorylation at Ser-174 and Ser-175 by the nuclear isoform of CK1. Mutation of eIF6 at the phosphorylatable Ser-174 and Ser-175 to alanine or treatment of cells with the CK1 inhibitor, D4476 inhibits nuclear export of eIF6 and results in nuclear accumulation of eIF6. Together, these results establish eIF6 as a substrate for calcineurin and suggest a novel paradigm for calcineurin function in 60 S ribosome biogenesis via regulating the nuclear accumulation of eIF6.

Tethering function of the caspase cleavage fragment of Golgi protein p115 promotes apoptosis via a p53-dependent pathway.

The Golgi apparatus undergoes extensive fragmentation during apoptosis due in part to caspase-mediated cleavage of its structural proteins. Significantly, the Golgi-vesicle-tethering protein p115 is cleaved at Asp(757) early during apoptosis and the nuclear translocation of its 205 amino acid C-terminal fragment (CTF) precedes observable Golgi fragmentation. Nuclear localization of the p115 CTF induces apoptosis. The regulation of CTF nuclear translocation and the mechanism of its apoptotic activity however, remain unknown. Here, we demonstrate that nuclear translocation of the CTF is regulated by SUMOylation. CTF-induced apoptosis is transcription dependent and mediated by the tumor suppressor, p53. Expression of the CTF led to the phosphorylation and stabilization of p53 and results in the expression of PUMA, a pro-apoptotic target of p53. CTF-induced stabilization of p53 is sensitive to the MEK/ERK inhibitor U0126. Co-immunoprecipitation studies indicate that the p115 CTF can bind to both p53 and ERK1. The CTF is also able to form dimers and its dimerization is dependent on residues 859-884, previously determined to be required for apoptosis. Indeed, CTF expression promotes p53-ERK interaction, which is diminished upon deletion of residues 859-884. Together, our results indicate a conserved tethering function of the Golgi protein p115 CTF which promotes p53-ERK interaction for the amplification of the apoptotic signal.

Phospholipase D in the Golgi apparatus.

Phospholipase D has long been implicated in vesicle formation and vesicular transport through the secretory pathway. The Golgi apparatus has been shown to exhibit a plethora of mechanisms of vesicle formation at different stages to accommodate a wide variety of cargo. Phospholipase D has been found on the Golgi apparatus and is regulated by ADP-ribosylation factors which are themselves regulators of vesicle trafficking. Moreover, the product of phospholipase D activity, phosphatidic acid, as well as its degradation product diacylglycerol, have been implicated in vesicle fission and fusion events. Here we summarize recent advances in the understanding of the role of phospholipase D at the Golgi apparatus.

Nuclear import is required for the pro-apoptotic function of the Golgi protein p115.

During apoptosis the Golgi apparatus undergoes irreversible fragmentation. In part, this results from caspase-mediated cleavage of several high molecular weight coiled-coil proteins, termed golgins. These include GM130, golgin 160, and the Golgi vesicle tethering protein p115, whose caspase cleavage generates a C-terminal fragment (CTF) of 205 residues. Here we demonstrate that early during apoptosis, following the rapid cleavage of p115, endogenous CTF translocated to the cell nucleus and its nuclear import was required to enhance the apoptotic response. Expression of a series of deletion constructs identified a putative alpha-helical region of 26 amino acids, whose expression alone was sufficient to induce apoptosis; deletion of these 26 residues from the CTF diminished its proapoptotic activity. This region contains several potential SUMOylation sites and co-expression of SUMO together with the SUMO ligase, UBC9, resulted in SUMOylation of the p115 CTF. Significantly, when cells were treated with drugs that induce apoptosis, SUMOylation enhanced the efficiency of p115 cleavage and the kinetics of apoptosis. A construct in which a nuclear export signal was fused to the N terminus of p115 CTF accumulated in the cytoplasm and surprisingly, its expression did not induce apoptosis. In contrast, treatment of cells expressing this chimera with the antibiotic leptomycin induced its translocation into the nucleus and resulted in the concomitant induction of apoptosis. These results demonstrate that nuclear import of the p115 CTF is required for it to stimulate the apoptotic response and suggest that its mode of action is confined to the nucleus.

Caspase cleavage of phospholipase D1 in vitro alters its regulation and reveals a novel property of the "loop" region.

Phospholipase D (PLD) has been implicated in mediating vesicular transport, mitosis, differentiation and apoptosis. The product of PLD activity, phosphatidic acid (PA) has mitogenic potential and elevated PLD expression has been detected in many tumor cell lines. Several reports have demonstrated that distinct PLD domains regulate its activity and that truncated forms of PLD retain enzymatic activity. We hypothesized that during apoptosis caspase cleavage of PLDs could result in modification of their activities. To test this idea, we have used in vitro translation of PLD1 and PLD2 which generated active enzymes exhibiting properties mimicking those of the endogenous proteins. Here we demonstrate that PLD1 was rapidly cleaved in vitro by caspases-8, -3 and -7. In contrast, PLD2 cleavage was delayed and its activity was unaffected by incubation with caspase-3. Significantly, following caspase cleavage the response of PLD1 to regulatory stimuli was altered; it was no longer activated by PKC and instead exhibited an increased activity in response to small GTPases. Notably, this enhanced activity was due to cleavage of PLD1 in the "loop" domain, a region previously associated with negative regulatory function. Thus our data have identified a novel regulatory domain in PLD1.

Fragmentation of the Golgi apparatus: an early apoptotic event independent of the cytoskeleton.

The Golgi apparatus undergoes irreversible fragmentation during apoptosis, in part as a result of caspase-mediated cleavage of several Golgi-associated proteins. However, Golgi structure and orientation is also regulated by the cytoskeleton and cytoskeletal changes have been implicated in inducing apoptosis. Consequently, we have analyzed the role of actin filaments and microtubules in apoptotic Golgi fragmentation. We demonstrate that in Fas receptor-activated cells, fragmentation of the Golgi apparatus was an early event that coincided with release of cytochrome c from mitochondria. Significantly, Golgi fragmentation preceded major changes in the organization of both the actin cytoskeleton and microtubules. In staurosporine-treated cells, actin filament organization was rapidly disrupted; however, the Golgi apparatus maintained its juxtanuclear localization and underwent complete fragmentation only at later times. Attempts to stabilize actin filaments with jasplakinolide prior to treatment with staurosporine did not prevent Golgi fragmentation. Finally, in response to Fas receptor activation or staurosporine treatment the levels of beta-actin or alpha-tubulin remained unaltered, whereas several Golgi proteins, p115 and golgin-160, underwent caspase-mediated cleavage. Our data demonstrate that breakdown of the Golgi apparatus is an early event during apoptosis that occurs independently of major changes to the actin and tubulin cytoskeleton.

A role for clathrin in reassembly of the Golgi apparatus.

The Golgi apparatus is a highly dynamic organelle whose organization is maintained by a proteinaceous matrix, cytoskeletal components, and inositol phospholipids. In mammalian cells, disassembly of the organelle occurs reversibly at the onset of mitosis and irreversibly during apoptosis. Several pharmacological agents including nocodazole, brefeldin A (BFA), and primary alcohols (1-butanol) induce reversible fragmentation of the Golgi apparatus. To dissect the mechanism of Golgi reassembly, rat NRK and GH3 cells were treated with 1-butanol, BFA, or nocodazole. During washout of 1-butanol, clathrin, a ubiquitous coat protein implicated in vesicle traffic at the trans-Golgi network and plasma membrane, and abundant clathrin coated vesicles were recruited to the region of nascent Golgi cisternae. Knockdown of endogenous clathrin heavy chain showed that the Golgi apparatus failed to reform efficiently after BFA or 1-butanol removal. Instead, upon 1-butanol washout, it maintained a compact, tight morphology. Our results suggest that clathrin is required to reassemble fragmented Golgi elements. In addition, we show that after butanol treatment the Golgi apparatus reforms via an initial compact intermediate structure that is subsequently remodeled into the characteristic interphase lace-like morphology and that reassembly requires clathrin.

Phospholipase D1 corrects impaired betaAPP trafficking and neurite outgrowth in familial Alzheimer's disease-linked presenilin-1 mutant neurons.

Presenilins (PS1/PS2) regulate proteolysis of beta-amyloid precursor protein (betaAPP) and affect its intracellular trafficking. Here, we demonstrate that a PS1-interacting protein, phospholipase D1 (PLD1), affects intracellular trafficking of betaAPP. Overexpression of PLD1 in PS1wt cells promotes generation of betaAPP-containing vesicles from the trans-Golgi network. Conversely, inhibition of PLD1 activity by 1-butanol decreases betaAPP trafficking in both wt and PS1-deficient cells. The subcellular localization of PLD1 is altered, and PLD enzymatic activity is reduced in cells expressing familial Alzheimer's disease (FAD) PS1 mutations compared with PS1wt cells. Overexpression of wt, but not catalytically inactive, PLD1 increases budding of betaAPP-containing vesicles from the trans-Golgi network in FAD mutant cells. Surface delivery of betaAPP is also increased by PLD1 in these cells. The impaired neurite outgrowth capacity in FAD mutant neurons was corrected by introducing PLD1 into these cells. The results indicate that PLD1 may represent a therapeutic target for rescuing compromised neuronal function in AD.

"Slip, sliding away": phospholipase D and the Golgi apparatus.

Phospholipase D enzymes (PLDs) constitute a family of phosphodiesterases that catalyze the hydrolysis of phosphatidylcholine (PtdCho) to generate choline and phosphatidic acid (PtdOH), a potent lipid signaling molecule implicated in numerous physiological processes. Mammalian PLDs have been localized to multiple organelles, including the nucleus, Golgi apparatus, lysosomes, secretory granules and plasma membrane. However, the detailed mechanisms that govern targeting of PLDs to different organelles, how their local activity is controlled or indeed the nature of PA effectors are not well understood. Here, we discuss recent observations on PLD localization to the Golgi apparatus and how members of this enzyme family might play a role in regulating the structure of this organelle.

Fragmentation of the Golgi apparatus. A role for beta III spectrin and synthesis of phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) synthesis has been implicated in maintaining the function of the Golgi apparatus. Here we demonstrate that the inhibition of PtdIns(4,5)P(2) synthesis in vitro in response to primary alcohol treatment and the kinetics of Golgi fragmentation in vivo were very rapid and tightly coupled. Preloading Golgi membranes with short chain phosphatidic acid abrogated the alcohol-mediated inhibition of PtdIns(4,5)P(2) synthesis in vitro. We also show that fragmentation of the Golgi apparatus in response to diminished PtdIns(4,5)P(2) synthesis correlated with both the phosphorylation of a Golgi form of beta III spectrin, a PtdIns(4,5)P(2)-interacting protein, and changes in its intracellular redistribution. The data are consistent with a model suggesting that the decreased PtdIns(4,5)P(2) synthesis and the phosphorylation state of beta III spectrin modulate the structural integrity of the Golgi apparatus.

Phospholipase D2 is localized to the rims of the Golgi apparatus in mammalian cells.

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid, a molecule known to have multiple physiological roles, including release of nascent secretory vesicles from the trans-Golgi network. In mammalian cells two forms of the enzyme, PLD1 and PLD2, have been described. We recently demonstrated that PLD1 is localized to the Golgi apparatus, nuclei, and to a lesser extent, plasma membrane. Due to its low abundance, the intracellular localization of PLD2 has been characterized only indirectly through overexpression of chimeric proteins. Using antibodies specific to PLD2, together with immunofluorescence microscopy, herein we demonstrate that a significant fraction of endogenous PLD2 localized to the perinuclear Golgi region and was also distributed throughout cells in dense cytoplasmic puncta; a fraction of which colocalized with caveolin-1 and the plasma membrane. On treatment with brefeldin A, PLD2 translocated into the nucleus in a manner similar to PLD1, suggesting a potential role in nuclear signaling. Most significantly, cryoimmunogold electron microscopy demonstrated that in pituitary GH(3) cells >90% of PLD2 present in the Golgi apparatus was localized to cisternal rims and peri-Golgi vesicles exclusively. The data are consistent with a model whereby PLD2 plays a role in Golgi vesicular transport.

A caspase cleavage fragment of p115 induces fragmentation of the Golgi apparatus and apoptosis.

In mammalian cells, the Golgi apparatus undergoes extensive fragmentation during apoptosis. p115 is a key vesicle tethering protein required for maintaining the structural organization of the Golgi apparatus. Here, we demonstrate that p115 was cleaved during apoptosis by caspases 3 and 8. Compared with control cells expressing native p115, those expressing a cleavage-resistant form of p115 delayed Golgi fragmentation during apoptosis. Expression of cDNAs encoding full-length or an NH2-terminal caspase cleavage fragment of p115 had no effect on Golgi morphology. In contrast, expression of the COOH-terminal caspase cleavage product of p115 itself caused Golgi fragmentation. Furthermore, this fragment translocated to the nucleus and its expression was sufficient to induce apoptosis. Most significantly, in vivo expression of the COOH-terminal fragment in the presence of caspase inhibitors, or upon coexpression with a cleavage-resistant mutant of p115, showed that p115 degradation plays a key role in amplifying the apoptotic response independently of Golgi fragmentation.

Evidence of a novel dipeptidyl aminopeptidase in mammalian GH(3) cells: new insights into the processing of peptide hormone precursors.

We investigated whether yeast signals could regulate hormone processing in mammalian cells. Chmeric genes coding for the prepro region of yeast alpha-factor and the functional hormone region of anglerfish somatostatin was expressed in rat pituitary GH(3) cells. The nascent prepro-alpha-factor-somatostatin peptides disappeared from cells with a half-life of 30 min, and about 20% of unprocessed precursors remained intracellular after a 2 h chase period. Disappearance of propeptide was insensitive to lysosomotropic agents, but was inhibited at 15 degrees C or 20 degrees C, suggesting that the hybrid propeptides were not degraded in the secretory pathway to the trans Golgi network or in lysosomes. It appeared that while most unprocessed precursors were constitutively secreted into the medium, a small portion were processed at their paired dibasic sites by prohormone-processing enzymes located in trans Golgi network/secretory vesicles, resulting in the production of mature somatostatin peptides. To test this hypothesis, we investigated the processing pattern of two different hybrid precursors: the 52-1 hybrid precursor, which has a Glu-Ala spacer between the prepro region of alpha-factor and somatostatin, and the 58-1 hybrid precursor, which lacks the Glu-Ala spacer. Processing of metabolically labeled hybrid propeptides to smaller somatostatin peptides was assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, 68% of the initially synthesized propeptides were secreted constitutively. About 22% of somatostatin-related products were proteolytically processed to mature somatostatin, of which 38.7% were detected intracellularly after 2 h. From N-terminal peptide sequence determination of somatostatin-related products in GH(3)-52 and GH(3)-58 cells, we found that both hybrid precursors were accurately cleaved at their dibasic amino acid sites. Notably, we also observed that the Glu-Ala spacer sequence was removed from 52-1 hybrid precursors. The latter result strongly suggests that a novel dipeptidyl aminopeptidase activity - a yeast STE13-like enzyme - is present in the post-trans Golgi network compartment of GH(3) cells. The data from these studies indicate that mechanisms which control protein secretion are conserved between yeast and mammalian cells.

Fragmentation and re-assembly of the Golgi apparatus in vitro. A requirement for phosphatidic acid and phosphatidylinositol 4,5-bisphosphate synthesis.

Recent work from our laboratory demonstrated that phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), are required to maintain the structural integrity of the Golgi apparatus. To investigate the role of these lipids in regulating Golgi structure and function, we developed a novel assay to follow the release of post-Golgi vesicles. Isolated rat liver Golgi membranes were incubated with [(3)H]CMP sialic acid to radiolabel endogenous soluble and membrane glycoproteins present in the late Golgi and trans-Golgi network. The release of post-Golgi secretory vesicles was determined by measuring incorporation of (3)H-labeled proteins into a medium speed supernatant. Vesicle budding was dependent on temperature, cytosol, energy and time. Electron microscopy of Golgi fractions prior to and after incubation demonstrated that the stacked Golgi cisternae generated a heterogeneous population of vesicles (50- to 350-nm diameter). Inhibition of phospholipase D-mediated PA synthesis, by incubation with 1-butanol, resulted in the complete fragmentation of the Golgi membranes in vitro into 50- to 100-nm vesicles; this correlated with diminished PtdIns(4,5)P(2) synthesis. Following alcohol washout, PA synthesis resumed and in the presence of cytosol PtdIns(4,5)P(2) synthesis was restored. Most significantly, under these conditions the fragmented Golgi elements reformed into flattened cisternae and the re-assembled Golgi supported vesicle release. These data demonstrate that inositol phospholipid synthesis is essential for the structure and function of the Golgi apparatus.

Intracellular trafficking and metabolic turnover of yeast prepro-alpha-factor-SRIF precursors in GH3 cells.

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that approximately 68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of approximately 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.