The Export of Major Histocompatibility Complex Class I Molecules from the Endoplasmic Reticulum of Rat Brown Adipose Cells Is Acutely Stimulated by Insulin

Major histocompatibility complex class I (MHC-I) molecules have been implicated in several nonimmunological functions including the regulation and intracellular trafficking of the insulin-responsive glucose transporter GLUT4. We have used confocal microscopy to compare the effects of insulin on the intracellular trafficking of MHC-I and GLUT4 in freshly isolated rat brown adipose cells. We also used a recombinant vaccinia virus (rVV) to express influenza virus hemagglutinin (HA) as a generic integral membrane glycoprotein to distinguish global versus specific enhancement of protein export from the endoplasmic reticulum (ER) in response to insulin. In the absence of insulin, MHC-I molecules largely colocalize with the ER-resident protein calnexin and remain distinct from intracellular pools of GLUT4. Surprisingly, insulin induces the rapid export of MHC-I molecules from the ER with a concomitant approximately three-fold increase in their level on the cell surface. This ER export is blocked by brefeldin A and wortmannin but is unaffected by cytochalasin D, indicating that insulin stimulates the rapid transport of MHC-I molecules from the ER to the plasma membrane via the Golgi complex in a phosphatidyl-inositol 3-kinase–dependent and actin-independent manner. We further show that the effect of insulin on MHC-I molecules is selective, because insulin does not affect the intracellular distribution or cell-surface localization of rVV-expressed HA. These results demonstrate that in rat brown adipose cells MHC-I molecule export from the ER is stimulated by insulin and provide the first evidence that the trafficking of MHC-I molecules is acutely regulated by a hormone.

Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein

Differential compartmentalization of signaling molecules in cells and tissues is being recognized as an important mechanism for regulating the specificity of signal transduction pathways. A kinase anchoring proteins (AKAPs) direct the subcellular localization of protein kinase A (PKA) by binding to its regulatory (R) subunits. Dual specific AKAPs (D-AKAPs) interact with both RI and RII. A 372-residue fragment of mouse D-AKAP2 with a 40-residue C-terminal PKA binding region and a putative regulator of G protein signaling (RGS) domain was previously identified by means of a yeast two-hybrid screen. Here, we report the cloning of full-length human D-AKAP2 (662 residues) with an additional putative RGS domain, and the corresponding mouse protein less the first two exons (617 residues). Expression of D-AKAP2 was characterized by using mouse tissue extracts. Full-length D-AKAP2 from various tissues shows different molecular weights, possibly because of alternative splicing or posttranslational modifications. The cloned human gene product has a molecular weight similar to one of the prominent mouse proteins. In vivo association of D-AKAP2 with PKA in mouse brain was demonstrated by using cAMP agarose pull-down assay. Subcellular localization for endogenous mouse, rat, and human D-AKAP2 was determined by immunocytochemistry, immunohistochemistry, and tissue fractionation. D-AKAP2 from all three species is highly enriched in mitochondria. The mitochondrial localization and the presence of RGS domains in D-AKAP2 may have important implications for its function in PKA and G protein signal transduction.

Vps10p Transport from the trans-Golgi Network to the Endosome Is Mediated by Clathrin-coated Vesicles

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12Δ and vps34Δ, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10CtΔp mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.

Phosphatidylcholine Synthesis Influences the Diacylglycerol Homeostasis Required for Sec14p-dependent Golgi Function and Cell Growth

Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes. In yeast, diacylglycerol accepts a phosphocholine moiety through a CPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine. EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo. In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway. Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells from SEC14-mediated cell death. The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20–40-fold over endogenous long-chain diacylglycerol levels. Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid.

Molecular Cloning and Characterization of Phocein, a Protein Found from the Golgi Complex to Dendritic Spines

Phocein is a widely expressed, highly conserved intracellular protein of 225 amino acids, the sequence of which has limited homology to the ς subunits from clathrin adaptor complexes and contains an additional stretch bearing a putative SH3-binding domain. This sequence is evolutionarily very conserved (80% identity between Drosophila melanogaster and human). Phocein was discovered by a yeast two-hybrid screen using striatin as a bait. Striatin, SG2NA, and zinedin, the three mammalian members of the striatin family, are multimodular, WD-repeat, and calmodulin-binding proteins. The interaction of phocein with striatin, SG2NA, and zinedin was validated in vitro by coimmunoprecipitation and pull-down experiments. Fractionation of brain and HeLa cells showed that phocein is associated with membranes, as well as present in the cytosol where it behaves as a protein complex. The molecular interaction between SG2NA and phocein was confirmed by their in vivo colocalization, as observed in HeLa cells where antibodies directed against either phocein or SG2NA immunostained the Golgi complex. A 2-min brefeldin A treatment of HeLa cells induced the redistribution of both proteins. Immunocytochemical studies of adult rat brain sections showed that phocein reactivity, present in many types of neurons, is strictly somato-dendritic and extends down to spines, just as do striatin and SG2NA.

Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle

We present evidence that a bacterial signal transduction cascade that couples morphogenesis with cell cycle progression is regulated by dynamic localization of its components. Previous studies have implicated two histidine kinases, DivJ and PleC, and the response regulator, DivK, in the regulation of morphogenesis in the dimorphic bacterium Caulobacter crescentus. Here, we show that the cytoplasmic response regulator, DivK, exhibits a dynamic, cyclical localization that culminates in asymmetric distribution of DivK within the two cell types that are characteristic of the Caulobacter cell cycle; DivK is dispersed throughout the cytoplasm of the progeny swarmer cell and is localized to the pole of the stalked cell. The membrane-bound DivJ and PleC histidine kinases, which are asymmetrically localized at the opposite poles of the predivisional cell, control the temporal and spatial localization of DivK. DivJ mediates DivK targeting to the poles whereas PleC controls its release from one of the poles at times and places that are consistent with the activities and location of DivJ and PleC in the late predivisional cell. Thus, dynamic changes in subcellular location of multiple components of a signal transduction cascade may constitute a novel mode of prokaryotic regulation to generate and maintain cellular asymmetry.

Localization of CD4+ T cell epitope hotspots to exposed strands of HIV envelope glycoprotein suggests structural influences on antigen processing

The spectrum of immunogenic epitopes presented by the H2-IAb MHC class II molecule to CD4+ T cells has been defined for two different (clade B and clade D) HIV envelope (gp140) glycoproteins. Hybridoma T cell lines were generated from mice immunized by a sequential prime and boost regime with DNA, recombinant vaccinia viruses, and protein. The epitopes recognized by reactive T cell hybridomas then were characterized with overlapping peptides synthesized to span the entire gp140 sequence. Evidence of clonality also was assessed with antibodies to T cell receptor Vα and Vβ chains. A total of 80 unique clonotypes were characterized from six individual mice. Immunogenic peptides were identified within only four regions of the HIV envelope. These epitope hotspots comprised relatively short sequences (≈20–80 aa in length) that were generally bordered by regions of heavy glycosylation. Analysis in the context of the gp120 crystal structure showed a pattern of uniform distribution to exposed, nonhelical strands of the protein. A likely explanation is that the physical location of the peptide within the native protein leads to differential antigen processing and consequent epitope selection.

Characterization of Chlamydomonas reinhardtii Zygote-Specific cDNAs That Encode Novel Proteins Containing Ankyrin Repeats and WW Domains1

Genes that are expressed only in the young zygote are considered to be of great importance in the development of an isogamous green alga, Chlamydomonas reinhardtii. Clones representing the Zys3 gene were isolated from a cDNA library prepared using zygotes at 10 min after fertilization. Sequencing of Zys3 cDNA clones resulted in the isolation of two related molecular species. One of them encoded a protein that contained two kinds of protein-to-protein interaction motifs known as ankyrin repeats and WW domains. The other clone lacked the ankyrin repeats but was otherwise identical. These mRNA species began to accumulate simultaneously in cells beginning 10 min after fertilization, and reached maximum levels at about 4 h, after which time levels decreased markedly. Genomic DNA gel-blot analysis indicated that Zys3 was a single-copy gene. The Zys3 proteins exhibited parallel expression to the Zys3 mRNAs at first, appearing 2 h after mating, and reached maximum levels at more than 6 h, but persisted to at least 1 d. Immunocytochemical analysis revealed their localization in the endoplasmic reticulum, which suggests a role in the morphological changes of the endoplasmic reticulum or in the synthesis and transport of proteins to the Golgi apparatus or related vesicles.

Structure, Properties, and Tissue Localization of Apoplastic α-Glucosidase in Crucifers1

Apoplastic α-glucosidases occur widely in plants but their function is unknown because appropriate substrates in the apoplast have not been identified. Arabidopsis contains at least three α-glucosidase genes; Aglu-1 and Aglu-3 are sequenced and Aglu-2 is known from six expressed sequence tags. Antibodies raised to a portion of Aglu-1 expressed in Escherichia coli recognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli (Brassica oleracea L.), and mustard (Brassica napus L.). The acidic α-glucosidase activity from broccoli flower buds was purified using concanavalin A and ion-exchange chromatography. Two active fractions were resolved and both contained a 96-kD immunoreactive polypeptide. The N-terminal sequence from the 96-kD broccoli α-glucosidase indicated that it corresponds to the Arabidopsis Aglu-2 gene and that approximately 15 kD of the predicted N terminus was cleaved. The 81-kD protein was more abundant than the 96-kD protein, but it was not active with 4-methylumbelliferyl-α-d-glucopyranoside as the substrate and it did not bind to concanavalin A. In situ activity staining using 5-bromo-4-chloro-3-indolyl-α-d-glucopyranoside revealed that the acidic α-glucosidase activity is predominantly located in the outer cortex of broccoli stems and in vascular tissue, especially in leaf traces.

Quantitative Intercellular Localization of NADH-Dependent Glutamate Synthase Protein in Different Types of Root Cells in Rice Plants1

The quantitative analysis with immunogold-electron microscopy using a single-affinity-purified anti-NADH-glutamate synthase (GOGAT) immunoglobulin G (IgG) as the primary antibody showed that the NADH-GOGAT protein was present in various forms of plastids in the cells of the epidermis and exodermis, in the cortex parenchyma, and in the vascular parenchyma of root tips (<10 mm) of rice (Oryza sativa) seedlings supplied with 1 mm NH4+ for 24 h. The values of the mean immunolabeling density of plastids were almost equal among these different cell types in the roots. However, the number of plastids per individual cell type was not identical, and some parts of the cells in the epidermis and exodermis contained large numbers of plastids that were heavily immunolabeled. Although there was an indication of labeling in the mitochondria using the single-affinity-purified anti-NADH-GOGAT IgG, this was not confirmed when a twice-affinity-purified IgG was used, indicating an exclusively plastidial location of the NADH-GOGAT protein in rice roots. These results, together with previous work from our laboratory (K. Ishiyama, T. Hayakawa, and T. Yamaya [1998] Planta 204: 288–294), suggest that the assimilation of exogeneously supplied NH4+ ions is primarily via the cytosolic glutamine synthetase/plastidial NADH-GOGAT cycle in specific regions of the epidermis and exodermis in rice roots. We also discuss the role of the NADH-GOGAT protein in vascular parenchyma cells.

Organellar and Cytosolic Localization of Four Phosphoribosyl Diphosphate Synthase Isozymes in Spinach

Four cDNAs encoding phosphoribosyl diphosphate (PRPP) synthase were isolated from a spinach (Spinacia oleracea) cDNA library by complementation of an Escherichia coli Δprs mutation. The four gene products produced PRPP in vitro from ATP and ribose-5-phosphate. Two of the enzymes (isozymes 1 and 2) required inorganic phosphate for activity, whereas the others were phosphate independent. PRPP synthase isozymes 2 and 3 contained 76 and 87 amino acid extensions, respectively, at their N-terminal ends in comparison with other PRPP synthases. Isozyme 2 was synthesized in vitro and shown to be imported and processed by pea (Pisum sativum) chloroplasts. Amino acid sequence analysis indicated that isozyme 3 may be transported to mitochondria and that isozyme 4 may be located in the cytosol. The deduced amino acid sequences of isozymes 1 and 2 and isozymes 3 and 4 were 88% and 75% identical, respectively. In contrast, the amino acid identities of PRPP synthase isozyme 1 or 2 with 3 or 4 was modest (22%–25%), but the sequence motifs for binding of PRPP and divalent cation-nucleotide were identified in all four sequences. The results indicate that PRPP synthase isozymes 3 and 4 belong to a new class of PRPP synthases that may be specific to plants.

The Schizosaccharomyces pombe spo20+ Gene Encoding a Homologue of Saccharomyces cerevisiae Sec14 Plays an Important Role in Forespore Membrane Formation

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20+ is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20+ gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

The ADP Ribosylation Factor-Nucleotide Exchange Factors Gea1p and Gea2p Have Overlapping, but Not Redundant Functions in Retrograde Transport from the Golgi to the Endoplasmic Reticulum

The activation of the small ras-like GTPase Arf1p requires the action of guanine nucleotide exchange factors. Four Arf1p guanine nucleotide exchange factors have been identified in yeast: Sec7p, Syt1p, Gea1p, and its homologue Gea2p. We identified GEA2 as a multicopy suppressor of a sec21-3 temperature-sensitive mutant. SEC21 encodes the γ-subunit of coatomer, a heptameric protein complex that together with Arf1p forms the COPI coat. GEA1 and GEA2 have at least partially overlapping functions, because deletion of either gene results in no obvious phenotype, whereas the double null mutant is inviable. Conditional mutants defective in both GEA1 and GEA2 accumulate endoplasmic reticulum and Golgi membranes under restrictive conditions. The two genes do not serve completely overlapping functions because a Δgea1 Δarf1 mutant is not more sickly than a Δarf1 strain, whereas Δgea2 Δarf1 is inviable. Biochemical experiments revealed similar distributions and activities for the two proteins. Gea1p and Gea2p exist both in membrane-bound and in soluble forms. The membrane-bound forms, at least one of which, Gea2p, can be visualized on Golgi structures, are both required for vesicle budding and protein transport from the Golgi to the endoplasmic reticulum. In contrast, Sec7p, which is required for protein transport within the Golgi, is not required for retrograde protein trafficking.

Evidence for an Intrinsic Toxicity of Phosphatidylcholine to Sec14p-dependent Protein Transport from the Yeast Golgi Complex

Yeast phosphatidylinositol-transfer protein (Sec14p) is essential for Golgi secretory function and cell viability. This requirement of Sec14p is relieved by genetic inactivation of the cytidine diphosphate-choline pathway for phosphatidycholine (PtdCho) biosynthesis. Standard phenotypic analyses indicate that inactivation of the phosphatidylethanolamine (PtdEtn) pathway for PtdCho biosynthesis, however, does not rescue the growth and secretory defects associated with Sec14p deficiency. We now report inhibition of choline uptake from the media reveals an efficient “bypass Sec14p” phenotype associated with PtdEtn-methylation pathway defects. We further show that the bypass Sec14p phenotype associated with PtdEtn-methylation pathway defects resembles other bypass Sec14p mutations in its dependence on phospholipase D activity. Finally, we find that increased dosage of enzymes that catalyze phospholipase D-independent turnover of PtdCho, via mechanisms that do not result in a direct production of phosphatidic acid or diacylglycerol, effect a partial rescue of sec14-1ts-associated growth defects. Taken together, these data support the idea that PtdCho is intrinsically toxic to yeast Golgi secretory function.