Identification of a glucose-6-phosphate isomerase involved in adaptation to salt stress of <Emphasis Type="Italic">Dunaliella salina</Emphasis>

The unicellular green alga Dunaliella salina is a recognized model for studying plant adaptation to high salinity. To isolate some salt-induced proteins at proteomics levels and to identify their expressions at gene levels, algal cells at logarithmic phase cultured in 1.5 and 3.5 M NaCl media were harvested for protein extraction. Solubilized proteins were applied to two-dimensional gel electrophoresis (2-DE) and analyzed by ImageMaster 2D Platinum software. Twenty-one protein spots whose intensities were elevated threefold to 13-fold at 3.5 M NaCl as compared to 1.5 M NaCl were analyzed by matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry. One salt-induced protein isolated from the 2-DE gels was identified as a glucose-6-phosphate isomerase (GPI) from D. salina (DsGPI). A full-length cDNA of DsGPI was obtained using rapid amplification of cDNA end technique, and it was shown by heterologous expression to encode a protein with a molecular weight consistent with the protein spot in the 2-DE gels. Real-time quantitative RT-PCR demonstrated that the mRNA of DsGPI was induced up to eightfold (P < 0.01) by 2.5 M and 14-fold higher (P < 0.01) by 3.5 M NaCl than by 1.5 M NaCl, respectively. It is concluded that the protein isolated through 2-DE is indeed DsGPI and that the DsGPI gene may be involved in adaptation to high salinity.     

A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes

Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein–Raf1 and the Raichu-KRas probe, we identified for the first time in vivo–active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14–MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell.     

mRNA expression of the murine glycoprotein (transmembrane) nmb (Gpnmb) gene is linked to the developing retinal pigment epithelium and iris

The murine homologue of the human glycoprotein (transmembrane) NMB (GPNMB) gene was identified by subtractive cloning from in vitro cultured murine primary osteoblast cells and subsequent RACE-PCR. GPNMB is a highly glycosylated type I transmembrane protein that shares significant sequence homology to several melanosomal proteins. Increasing expression of Gpnmb mRNA was observed during differentiation of murine primary osteoblast cell cultures. To address the potential functions of GPNMB we analysed its mRNA-expression during murine embryonic development. In early development Gpnmb mRNA is detected at high levels in the outer layer of the retina. Later in development expression gets restricted to the retinal pigment epithelium and iris. At the cytoplasmic domain of GPNMB, a conserved di-leucin-based endosomal/melanosomal-sorting signal (ExxPLL) was located, present as well in several known melanosomal proteins. To analyse the subcellular localization we used EGFP-tagged GPNMB transfected in COS7 and HEK293 cells. In both non-pigmented cell lines, the EGFP-GPNMB fusion protein was localized to vesicular, endosomal like structures. Sequence homology to known melanosomal proteins, mRNA expression and subcellular localization are suggestive for GPNMB as an intracellular, endosomal/melanosomal compartment specific protein important for melanin biosynthesis and the development of the retinal pigment epithelium and iris. As the gene coding for human GPNMB was localized to chromosome 7p15, a locus involved in the human inherited disease cystoid macular edema, also known as dominant cystoid macular dystrophy (OMIM 153880) we highly suggest that GPNMB is a candidate gene for this human inherited disease.     

Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2^−/− fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment.     

Expression of the spr1 gene in cultured tracheal epithelial cells and its regulation by retinoids before and after confluence

The absence of vitamin A or vitamin A derivatives in culture media promotes squamous cell differentiation of tracheobronchial epithelial cells. This is especially true for the expression of a small proline-rich protein (20K; 98 amino acids) in pig trachea epithelial cells. Multigene families encode different small proline-rich proteins in different species, and these proteins are possible markers for squamous cell differentiation. 20K mRNA and 20K protein were detected in cells within 4 and 5 days in culture, respectively, when cells reached about 50% confluence, and expression increased 12-fold during cell proliferation until cells reached 100% confluence. Arotinoid (10⁻⁹ M), a synthetic retinoid, essentially totally inhibited expression of 20K mRNA in proliferating tracheobronchial cells within 3 days of treatment while 20K protein levels were only decreased 4-fold after 5 days. However, if cells were exposed to arotinoid 3 days after reaching confluent growth, the levels of either 20K mRNA or 20K protein were unchanged. Cells exposed to arotinoid from the onset of culturing, and then removal of the retinoid from proliferating cells resulted in the expression of 20K mRNA and protein after 4 and 5 days as observed previously. 20K mRNA was not detected in cells that had been continuously exposed to arotinoid from the start of culture until 3 days post confluence, even 10 days following removal of arotinoid. Our results strongly suggest that the growth phase and state of cell differentiation greatly affect the response of these epithelial cells to vitamin A derivatives. © 1996 Wiley-Liss, Inc.     

Endosomal cargo recycling mediated by Gpa1 and phosphatidylinositol 3-kinase is inhibited by glucose starvation

Cell surface protein trafficking is regulated in response to nutrient availability, with multiple pathways directing surface membrane proteins to the lysosome for degradation in response to suboptimal extracellular nutrients. Internalized protein and lipid cargoes recycle back to the surface efficiently in glucose-replete conditions, but this trafficking is attenuated following glucose starvation. We find that cells with either reduced or hyperactive phosphatidylinositol 3-kinase (PI3K) activity are defective for endosome to surface recycling. Furthermore, we find that the yeast Gα subunit Gpa1, an endosomal PI3K effector, is required for surface recycling of cargoes. Following glucose starvation, mRNA and protein levels of a distinct Gα subunit Gpa2 are elevated following nuclear translocation of Mig1, which inhibits recycling of various cargoes. As Gpa1 and Gpa2 interact at the surface where Gpa2 concentrates during glucose starvation, we propose that this disrupts PI3K activity required for recycling, potentially diverting Gpa1 to the surface and interfering with its endosomal role in recycling. In support of this model, glucose starvation and overexpression of Gpa2 alter PI3K endosomal phosphoinositide production. Glucose deprivation therefore triggers a survival mechanism to increase retention of surface cargoes in endosomes and promote their lysosomal degradation.     

Biogenesis of lysosome‐related organelles complex‐1 (BORC) regulates late endosomal/lysosomal size through PIKfyve‐dependent phosphatidylinositol‐3,5‐bisphosphate

Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC‐1‐related complex (BORC) regulates the size of these organelles via PIKfyve‐dependent phosphatidylinositol‐3,5‐bisphosphate [PI(3,5)P₂] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P₂, suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP‐activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen‐activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.     

Decreased steady-state insulin-like growth factor binding protein-3 (IGFBP-3) mRNA level is associated with differentiation of cultured porcine myogenic cells

Insulin-like growth factor binding proteins (IGFBPs) affect the biological activity of IGF-I in several cell types, including cultured muscle cells. Additionally, at least one of the IGFBPs, IGFBP-3, has been shown to have IGF-independent effects on cell proliferation. Numerous studies have shown that immortalized muscle cell lines produce various IGFBPs, but to date no muscle cell line has been reported to produce IGFBP-3 protein or mRNA. Unlike muscle cell lines, primary cultures of porcine embryonic myogenic cells express IGFBP-3 mRNA and secrete a protein that is immunologically identifiable as IGFBP-3. Additionally, steady-state IGFBP-3 levels change significantly during differentiation. Here we report that differentiation of porcine myogenic cells in an IGFBP-3-free medium is accompanied by reduced steady-state IGFBP-3 mRNA levels. Steady-state levels of IGFBP-3 mRNA decreased approximately sevenfold (P < .05) during differentiation and then increased to predifferentiation levels once differentiation was complete. Addition of TGF-beta1 (0.5 ng/ml) to porcine myogenic cultures suppressed fusion and resulted in a sevenfold increase in steady-state IGFBP-3 mRNA and a 1.8-fold increase in IGFBP-3 protein levels as compared to untreated control cultures (P < .05). Results suggest that alterations in IGFBP-3 mRNA and protein may play a role in differentiation of porcine embryonic muscle cells.     

Cell Cycle-Specific Expression of G0SPR1 in Chinese Hamster Ovary Cells

A family of small proline-rich proteins (SPR1s) is induced in cells undergoing squamous differentiation. Because SPR1 mRNA is detected in mesenchymal nasal cells of rats exposed to cigarette smoke, expression of this mRNA in other nonsquamous cells and tissues was investigated. Using PCR, low levels of SPR1 mRNA were identified in a number of nondifferentiating cell lines and in nonsquamous tissues. G₀SPR1 mRNA, the hamster homologue of SPR1 mRNA, was increased 10-fold in Chinese hamster ovary (CHO) cells when the culture reached 80–90% confluence and was downregulated after cells ceased growing at 100% confluence. The deduced amino acid sequence of G₀SPR1 showed a high homology to the family of SPR1 from different species. Affinity-purified antibodies to SPR1 reacted to about 50% of the CHO cell population, indicating that the protein is expressed at specific stages of the cell cycle. CHO cells that were switched to low-serum medium when they were at 60% confluence showed an increase in G₀SPR1 levels before the cells entered G₀, indicating that G₀SPR1 may be a signal to cells entering G₀. Because expression of the SPR1 family of proteins is associated with squamous differentiation, the observations in the nondifferentiating CHO cells indicate that these proteins may play a role in mediating the withdrawal from the cell cycle prior to the commitment to differentiation.     

Roles of BLOC-1 and Adaptor Protein-3 Complexes in Cargo Sorting to Synaptic Vesicles

Neuronal lysosomes and their biogenesis mechanisms are primarily thought to clear metabolites and proteins whose abnormal accumulation leads to neurodegenerative disease pathology. However, it remains unknown whether lysosomal sorting mechanisms regulate the levels of membrane proteins within synaptic vesicles. Using high-resolution deconvolution microscopy, we identified early endosomal compartments where both selected synaptic vesicle and lysosomal membrane proteins coexist with the adaptor protein complex 3 (AP-3) in neuronal cells. From these early endosomes, both synaptic vesicle membrane proteins and characteristic AP-3 lysosomal cargoes can be similarly sorted to brain synaptic vesicles and PC12 synaptic-like microvesicles. Mouse knockouts for two Hermansky–Pudlak complexes involved in lysosomal biogenesis from early endosomes, the ubiquitous isoform of AP-3 (Ap3b1^−/−) and muted, defective in the biogenesis of lysosome-related organelles complex 1 (BLOC-1), increased the content of characteristic synaptic vesicle proteins and known AP-3 lysosomal proteins in isolated synaptic vesicle fractions. These phenotypes contrast with those of the mouse knockout for the neuronal AP-3 isoform involved in synaptic vesicle biogenesis (Ap3b2^−/−), in which the content of select proteins was reduced in synaptic vesicles. Our results demonstrate that lysosomal and lysosome-related organelle biogenesis mechanisms regulate steady-state synaptic vesicle protein composition from shared early endosomes.     

UBE4B Protein Couples Ubiquitination and Sorting Machineries to Enable Epidermal Growth Factor Receptor (EGFR) Degradation

The signaling of plasma membrane proteins is tuned by internalization and sorting in the endocytic pathway prior to recycling or degradation in lysosomes. Ubiquitin modification allows recognition and association of cargo with endosomally associated protein complexes, enabling sorting of proteins to be degraded from those to be recycled. The mechanism that provides coordination between the cellular machineries that mediate ubiquitination and endosomal sorting is unknown. We report that the ubiquitin ligase UBE4B is recruited to endosomes in response to epidermal growth factor receptor (EGFR) activation by binding to Hrs, a key component of endosomal sorting complex required for transport (ESCRT) 0. We identify the EGFR as a substrate for UBE4B, establish UBE4B as a regulator of EGFR degradation, and describe a mechanism by which UBE4B regulates endosomal sorting, affecting cellular levels of the EGFR and its downstream signaling. We propose a model in which the coordinated action of UBE4B, ESCRT-0, and the deubiquitinating enzyme USP8 enable the endosomal sorting and lysosomal degradation of the EGFR.