Membrane lipid perturbation modifies the set point of the temperature of heat shock response in yeast.

Addition of a saturated fatty acid (SFA) induced a strong increase in heat shock (HS) mRNA transcription when cells were heat-shocked at 37 degrees C, whereas treatment with an unsaturated fatty acid (UFA) reduced or eliminated the level of HS gene transcription at 37 degrees C. Transcription of the delta 9-desaturase gene (Ole1) of Histoplasma capsulatum, whose gene product is responsible for the synthesis of UFA, is up-regulated in a temperature-sensitive strain. We show that when the L8-14C mutant of Saccharomyces cerevisiae, which has a disrupted Ole1 gene, is complemented with its own Ole1 coding region under control of its own promoter or Ole1 promoters of H. capsulatum, the level of HS gene transcription depends on the activity of the promoters. Fluorescence anisotropy of mitochondrial membranes of completed strains corresponded to the different activity of the Ole1 promoter used. We propose that the SFA/UFA ratio and perturbation of membrane lipoprotein complexes are involved in the perception of rapid temperature changes and under HS conditions disturbance of the preexisting membrane physical state causes transduction of a signal that induces transcription of HS genes.

Glycoprotein 330/megalin: probable role in receptor-mediated transport of apolipoprotein J alone and in a complex with Alzheimer disease amyloid beta at the blood-brain and blood-cerebrospinal fluid barriers.

A soluble form of Alzheimer disease amyloid beta-protein (sA beta) is transported in the blood and cerebrospinal fluid mainly complexed with apolipoprotein J (apoJ). Using a well-characterized in situ perfused guinea pig brain model, we recently obtained preliminary evidence that apoJ facilitates transport of sA beta (1-40)-apoJ complexes across the blood-brain barrier and the blood-cerebrospinal fluid barrier, but the mechanisms remain poorly understood. In the present study, we examined the transport process in greater detail and investigated the possible role of glycoprotein 330 (gp330)/megalin, a receptor for multiple ligands, including apoJ. High-affinity transport systems with a Km of 0.2 and 0.5 nM were demonstrated for apoJ at the blood-brain barrier and the choroid epithelium in vivo, suggesting a specific receptor-mediated mechanism. The sA beta (1-40)-apoJ complex shared the same transport mechanism and exhibited 2.4- to 10.2-fold higher affinity than apoJ itself. Binding to microvessels, transport into brain parenchyma, and choroidal uptake of both apoJ and sA beta (1-40)-apoJ complexes were markedly inhibited (74-99%) in the presence of a monoclonal antibody to gp330/megalin and were virtually abolished by perfusion with the receptor-associated protein, which blocks binding of all known ligands to gp330. Western blot analysis of cerebral microvessels with the monoclonal antibody to gp330 revealed a protein with a mass identical to that in extracts of kidney membranes enriched with gp330/megalin, but in much lower concentration. The findings suggest that gp330/megalin mediates cellular uptake and transport of apoJ and sA beta (1-40)-apoJ complex at the cerebral vascular endothelium and choroid epithelium.

Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E.

The structure of m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin D1 and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NTH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin D1 and ODC. We show that the increase in the amount of cyclin D1 and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin D1 mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin D1 mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation.

Transport of cytoskeletal elements in the squid giant axon.

In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon.

Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes.

Signals for endocytosis and for basolateral and lysosomal sorting are closely related in a number of membrane proteins, suggesting similar sorting mechanisms at the plasma membrane and in the trans-Golgi network (TGN). We tested the hypothesis that basolateral membrane proteins are transported to the cell surface via endosomes for the asialoglycoprotein receptor H1. This protein was tagged with a tyrosine sulfation site (H1TS) to allow specific labeling with [35S]sulfate in the TGN. Madin-Darby canine kidney cells expressing H1TS were pulse-labeled and chased for a period of time insufficient for labeled H1TS to reach the cell surface. Upon homogenization and gradient centrifugation, fractions devoid of TGN were subjected to immunoisolation of compartments containing mannose 6-phosphate receptor, which served as an endosomal marker. H1TS in transit to the cell surface was efficiently coisolated, whereas a labeled secretory protein and free glycosaminoglycan chains were not. This indicates an indirect pathway for the asialoglycoprotein receptor to the plasma membrane via endosomes and has important implications for protein sorting in the TGN and endosomes.

Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult.

The recently cloned, distant member of the transforming growth factor beta (TGF-beta) family, glial cell line-derived neurotrophic factor (GDNF), has potent trophic actions on fetal mesencephalic dopamine neurons. GDNF also has protective and restorative activity on adult mesencephalic dopaminergic neurons and potently protects motoneurons from axotomy-induced cell death. However, evidence for a role for endogenous GDNF as a target-derived trophic factor in adult midbrain dopaminergic circuits requires documentation of specific transport from the sites of synthesis in the target areas to the nerve cell bodies themselves. Here, we demonstrate that GDNF is retrogradely transported by mesencephalic dopamine neurons of the nigrostriatal pathway. The pattern of retrograde transport following intrastriatal injections indicates that there may be subpopulations of neurons that are GDNF responsive. Retrograde axonal transport of biologically active 125I-labeled GDNF was inhibited by an excess of unlabeled GDNF but not by an excess of cytochrome c. Specificity was further documented by demonstrating that another TGF-beta family member, TGF-beta 1, did not appear to affect retrograde transport. Retrograde transport was also demonstrated by immunohistochemistry by using intrastriatal injections of unlabeled GDNF. GDNF immunoreactivity was found specifically in dopamine nerve cell bodies of the substantia nigra pars compacta distributed in granules in the soma and proximal dendrites. Our data implicate a specific receptor-mediated uptake mechanism operating in the adult. Taken together, the present findings suggest that GDNF acts endogenously as a target-derived physiological survival/maintenance factor for dopaminergic neurons.

Pharmacological modulation of heat shock factor 1 by antiinflammatory drugs results in protection against stress-induced cellular damage.

The activation of heat shock genes by diverse forms of environmental and physiological stress has been implicated in a number of human diseases, including ischemic damage, reperfusion injury, infection, neurodegeneration, and inflammation. The enhanced levels of heat shock proteins and molecular chaperones have broad cytoprotective effects against acute lethal exposures to stress. Here, we show that the potent antiinflammatory drug indomethacin activates the DNA-binding activity of human heat shock transcription factor 1 (HSF1). Perhaps relevant to its pharmacological use, indomethacin pretreatment lowers the temperature threshold of HSF1 activation, such that a complete heat shock response can be attained at temperatures that are by themselves insufficient. The synergistic effect of indomethacin and elevated temperature is biologically relevant and results in the protection of cells against exposure to cytotoxic conditions.

Suppression of heat-induced hsp70 expression by the 70-kDa subunit of the human Ku autoantigen.

Expression of the 70-kDa polypeptide of human Ku autoantigen in rat cells is shown to suppress specifically the induction of hsp70 upon heat shock. Thermal induction of other heat shock proteins is not significantly affected, nor is the state of phosphorylation or the DNA-binding ability of the heat shock transcription factor HSF1. These findings support a model in which hsp70 gene expression is controlled by a second regulatory factor in addition to the positive activator HSF1. The Ku autoantigen, or a protein closely related to it, is likely to be involved in the regulation of hsp70 expression.

Ammonia acquisition in enteric bacteria: Physiological role of the ammonium/methylammonium transport B (AmtB) protein

Homologues of the amtB gene of enteric bacteria exist in all three domains of life. Although their products are required for transport of the ammonium analogue methylammonium in washed cells, only in Saccharomyces cerevisiae have they been shown to be necessary for growth at low NH4+ concentrations. We now demonstrate that an amtB strain of Escherichia coli also grows slowly at low NH4+ concentrations in batch culture, but only at pH values below 7. In addition, we find that the growth defect of an S. cerevisiae triple-mutant strain lacking the function of three homologues of the ammonium/methylammonium transport B (AmtB) protein [called methylammonium/ammonium permeases (MEP)] that was observed at pH 6.1 is relieved at pH 7.1. These results provide direct evidence that AmtB participates in acquisition of NH4+/NH3 in bacteria as well as eucarya. Because NH3 is the species limiting at low pH for a given total concentration of NH4+ + NH3, results with both organisms indicate that AmtB/MEP proteins function in acquisition of the uncharged form. We confirmed that accumulation of [14C]methylammonium depends on its conversion to γ-N-methylglutamine, an energy-requiring reaction catalyzed by glutamine synthetase, and found that at pH 7, constitutive expression of AmtB did not relieve the growth defects of a mutant strain of Salmonella typhimurium that appears to require a high internal concentration of NH4+/NH3. Hence, contrary to previous views, we propose that AmtB/MEP proteins increase the rate of equilibration of the uncharged species, NH3, across the cytoplasmic membrane rather than actively transporting—that is, concentrating—the charged species, NH4+.

Uridine Diphosphate–Glucose Transport into the Endoplasmic Reticulum of Saccharomyces cerevisiae: In Vivo and In Vitro Evidence

It has been proposed that synthesis of β-1,6-glucan, one of Saccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose–dependent reaction in the lumen of the endoplasmic reticulum (ER). Because this sugar nucleotide is not synthesized in the lumen of the ER, we have examined whether or not UDP–glucose can be transported across the ER membrane. We have detected transport of this sugar nucleotide into the ER in vivo and into ER–containing microsomes in vitro. Experiments with ER-containing microsomes showed that transport of UDP–glucose was temperature dependent and saturable with an apparent Km of 46 μM and a Vmax of 200 pmol/mg protein/3 min. Transport was substrate specific because UDP–N-acetylglucosamine did not enter these vesicles. Demonstration of UDP–glucose transport into the ER lumen in vivo was accomplished by functional expression of Schizosaccharomyces pombe UDP–glucose:glycoprotein glucosyltransferase (GT) in S. cerevisiae, which is devoid of this activity. Monoglucosylated protein-linked oligosaccharides were detected in alg6 or alg5 mutant cells, which transfer Man9GlcNAc2 to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT. Deletion mutants, kre5Δ, lack cell wall β-1,6 glucan and grow very slowly. Expression of S. pombe GT in kre5Δ mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.

Actin Filaments and Microtubules are Involved in Different Membrane Traffic Pathways That Transport Sphingolipids to the Apical Surface of Polarized HepG2 Cells

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked “hyperpolarization,” i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements.

Shr3p Mediates Specific COPII Coatomer–Cargo Interactions Required for the Packaging of Amino Acid Permeases Into ER-derived Transport Vesicles

The SHR3 gene of Saccharomyces cerevisiae encodes an integral membrane component of the endoplasmic reticulum (ER) with four membrane-spanning segments and a hydrophilic, cytoplasmically oriented carboxyl-terminal domain. Mutations in SHR3 specifically impede the transport of all 18 members of the amino acid permease (aap) gene family away from the ER. Shr3p does not itself exit the ER. Aaps fully integrate into the ER membrane and fold properly independently of Shr3p. Shr3p physically associates with the general aap Gap1p but not Sec61p, Gal2p, or Pma1p in a complex that can be purified from N-dodecylmaltoside-solubilized membranes. Pulse–chase experiments indicate that the Shr3p–Gap1p association is transient, a reflection of the exit of Gap1p from the ER. The ER-derived vesicle COPII coatomer components Sec13p, Sec23p, Sec24p, and Sec31p but not Sar1p bind Shr3p via interactions with its carboxyl-terminal domain. The mutant shr3-23p, a nonfunctional membrane-associated protein, is unable to associate with aaps but retains the capacity to bind COPII components. The overexpression of either Shr3p or shr3-23p partially suppresses the temperature-sensitive sec12-1 allele. These results are consistent with a model in which Shr3p acts as a packaging chaperone that initiates ER-derived transport vesicle formation in the proximity of aaps by facilitating the membrane association and assembly of COPII coatomer components.

Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties

Bile secretion involves the structural and functional interplay of hepatocytes and cholangiocytes, the cells lining the intrahepatic bile ducts. Hepatocytes actively secrete bile acids into the canalicular space and cholangiocytes then transport bile acids in a vectorial manner across their apical and basolateral plasma membranes. The initial step in the transepithelial transport of bile acids across rat cholangiocytes is apical uptake by a Na+-dependent bile acid transporter (ASBT). To date, the molecular basis of the obligate efflux mechanism for extrusion of bile acids across the cholangiocyte basolateral membrane remains unknown. We have identified an exon-2 skipped, alternatively spliced form of ASBT, designated t-ASBT, expressed in rat cholangiocytes, ileum, and kidney. Alternative splicing causes a frameshift that produces a 154-aa protein. Antipeptide antibodies detected the ≈19 kDa t-ASBT polypeptide in rat cholangiocytes, ileum, and kidney. The t-ASBT was specifically localized to the basolateral domain of cholangiocytes. Transport studies in Xenopus oocytes revealed that t-ASBT can function as a bile acid efflux protein. Thus, alternative splicing changes the cellular targeting of ASBT, alters its functional properties, and provides a mechanism for rat cholangiocytes and other bile acid-transporting epithelia to extrude bile acids. Our work represents an example in which a single gene appears to encode via alternative splicing both uptake and obligate efflux carriers in a bile acid-transporting epithelial cell.

Evolutionary specialization of the nuclear targeting apparatus

The α- and β-karyopherins (Kaps), also called importins, mediate the nuclear transport of proteins. All α-Kaps contain a central domain composed of eight approximately 40 amino acid, tandemly arranged, armadillo-like (Arm) repeats. The number and order of these repeats have not changed since the common origin of fungi, plants, and mammals. Phylogenetic analysis suggests that the various α-Kaps fall into two groups, α1 and α2. Whereas animals encode both types, the yeast genome encodes only an α1-Kap. The β-Kaps are characterized by 14–15 tandemly arranged HEAT motifs. We show that the Arm repeats of α-Kaps and the HEAT motifs of β-Kaps are similar, suggesting that the α-Kaps and β-Kaps (and for that matter, all Arm and HEAT repeat-containing proteins) are members of the same protein superfamily. Phylogenetic analysis indicates that there are at least three major groups of β-Kaps, consistent with their proposed cargo specificities. We present a model in which an α-independent β-Kap progenitor gave rise to the α-dependent β-Kaps and the α-Kaps.

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide–MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAP-specific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis–Menten kinetics with a maximal velocity Vmax of 2 μmol/min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.