Molecular motors in axonal transport

Neurons require a large amount of intracellular transport. Cytoplasmic polypeptides and membrane-bounded organelles move from the perikaryon, down the length of the axon, and to the synaptic terminals. This movement occurs at distinct rates and is termed axonal transport. Axonal transport is divided into the slow transport of cytoplasmic proteins including glycolytic enzymes and cytoskeletal structures and the fast transport of membrane-bounded organelles along linear arrays of microtubules. The polypeptide compositions of the rate classes of axonal transport have been well characterized, but the underlying molecular mechanisms of this movement are less clear. Progress has been particularly slow toward understanding force-generation in slow transport, but recent developments have provided insight into the molecular motors involved in fast axonal transport. Recent advances in the cellular and molecular biology of one fast axonal transport motor, kinesin, have provided a clearer understanding of organelle movement along microtubules. The availability of cellular and molecular probes for kinesin and other putative axonal transport motors have led to a reevaluation of our understanding of intracellular motility.     

Naturally occurring virulence-attenuated isolates of Listeria monocytogenes capable of inducing long term protection against infection by virulent strains of homologous and heterologous serotypes

Abstract Experimental infections of mice with strains of Listeria spp. isolated from contaminated food sources allowed discrimination of strains into those either exhibiting high, attenuated or low virulence. Compared to the highly virulent L. monocytogenes strain EGD, an attenuated strain such as L99 persisted for shorter times (5 versus 10 days) in the infected host. Using a tissue culture cell model of infection, we found that, although strain L99 was capable of accumulatinn actin like its virulent counterpart following invasion, it was unable to generate the polarized actin tails required for intracellular and cell-to-cell movement. Immunoblot analysis using specific antiserum to the ActA polypeptide, a molecule that is necessary for movement of the bacterium within the eucaryotic cell, indicated that a slightly truncated form of this polypeptide was produced in the L99 strain. Despite its reduced virulence, the attenuated strain L99 was just as effective in generating protection in immune mice as the highly virulent strains, albeit with a 1000-fold higher infective dose. Based on the results obtained from this study, we suggest that one of the mechanisms accounting for widespread resistance in humans to infection by Listeria may be due to asymptomatic infections by naturally occurring strains attenuated for virulence.     

Ionic-strength-dependent changes in the structure of the major protein of the human erythrocyte membrane.

The effect of ionic strength on the proteolysis by trypsin of the major membrane-penetrating protein (polypeptide 3) in the erythrocyte membrane was studied. Both the intracellular and extracellular regions of the protein are susceptible to trypsin proteolysis under hypo-osmotic conditions, whereas under iso-osmotic conditions the extracellular region of the protein is resistant to trypsin, and the intracellular region yields only two cleavage products with trypsin. Studies of the fragments obtained from polypeptide 3 by trypsin digestion under iso-osmotic conditions of 'ghosts' radioiodinated with lactoperoxidase confirmed our earlier conclusions that the polypeptide chain of polypeptide 3 traverses the membrane twice. Ionic-strength-dependent changes were also observed in the incorporation of iodine by lactoperoxidase into the individual extracellular tyrosine sites of the protein. These results show that polypeptide 3 undergoes ionic-strength-dependent changes in structure.     

A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.     

Biosynthesis of alpha-fetoprotein in cultured hepatoma cells

Pulse and pulse-chase experiments demonstrated that a heterogeneous polypeptide with an apparent Mr = 68,000 was the first intracellular anti-alpha-fetoprotein (AFP)-precipitable polypeptide synthesized by rat Mc-A-RH-7777 hepatoma cells. The 68,000-dalton polypeptide may consist of polypeptides with apparent molecular weights ranging from 68,000 to 70,000. It was the precursor of two intracellular anti-AFP-precipitable polypeptides of 69,000 and 73,000 apparent molecular weight. The latter were secreted into the medium without further processing. The anti-AFP-precipitable polypeptides in both cells and medium incorporated [3H]glucosamine, indicating that these polypeptides are at least partially glycosylated. The 68,000-dalton polypeptide in cells was bound mostly to concanavalin A-Sepharose, whereas the 69,000-dalton polypeptide was entirely unbound. The 73,000-dalton polypeptide consisted of concanavalin A-bound and -unbound variants. Tunicamycin completely abolished the uptake of [3H]glucosamine into anti-AFT-precipitable polypeptides in both cells and medium, and the resulting polypeptide of apparent Mr = 66,000 did not bind to concanavalin A-Sepharose. Tunicamycin did not affect the synthesis or secretion of AFP by hepatoma cells.     

The mitochondrial protein import motor

Mitochondrial proteins are synthesized as precursor proteins in the cytosol and are posttranslationally imported into the organelle. A complex system of translocation machineries recognizes and transports the precursor polypeptide across the mitochondrial membranes. Energy for the translocation process is mainly supplied by the mitochondrial membrane potential (deltapsi) and the hydrolysis of ATP. Mitochondrial Hsp70 (mtHsp70) has been identified as the major ATPase driving the membrane transport of the precursor polypeptides into the mitochondrial matrix. Together with the partner proteins Tim44 and Mge1, mtHsp70 forms an import motor complex interacting with the incoming preproteins at the inner face of the inner membrane. This import motor complex drives the movement of the polypeptides in the translocation channel and the unfolding of carboxy-terminal parts of the preproteins on the outside of the outer membrane. Two models of the molecular mechanism of mtHsp70 during polypeptide translocation are discussed. In the 'trapping' model, precursor movement is generated by Brownian movement of the polypeptide chain in the translocation pore. This random movement is made vectorial by the interaction with mtHsp70 in the matrix. The detailed characterization of conditional mutants of the import motor complex provides the basis for an extended model. In this 'pulling' model, the attachment of mtHsp70 at the inner membrane via Tim44 and a conformational change induced by ATP results in the generation of an inward-directed force on the bound precursor polypeptide. This active role of the import motor complex is necessary for the translocation of proteins containing tightly folded domains. We suggest that both mechanisms complement each other to reach a high efficiency of preprotein import.     

Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root

Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosaccharide moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [³H]glucosamine and [³⁵S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.     

Dissociation of tetanus neurotoxin into two polypeptide fragments

Analyses of neurotoxin protein of $\text{Clostridium tetani}$ by polyacrylamide gel electrophoresis showed that the toxin as purified from culture filtrates (“extracellular” toxin, molecular weight 160,000) could be dissociated into two polypeptide chains of molecular weight 53,000 (Fragment α) and 107,000 (Fragment β) by treatment with dithiothreitol and sodium dodecyl sulfate. The toxin as purified from bacterial extracts (“intracellular” toxin) was found to consist of a single 160,000 dalton polypeptide chain, which is undissociable by such treatment but, when pretreated with trypsin, becomes dissociable into two fragments apparently identical with α and β.     

PKCzeta is required for microtubule-based motility of vesicles containing the ntcp transporter

Intracellular trafficking regulates the abundance and therefore activity of transporters present at the plasma membrane. The transporter, Na+-taurocholate co-transporting polypeptide (ntcp), is increased at the plasma membrane upon treatment of cells with cAMP, for which microtubules (MTs) are required and the PI3K pathway and PKCzeta have been implicated. However, trafficking of ntcp on MTs has not been demonstrated directly and the regulation and intracellular localization of ntcp is not well understood. Here, we utilize in vitro and whole-cell immunofluorescence microscopy assays to demonstrate that ntcp is present on intracellular vesicles that bind MTs and move bidirectionally, using kinesin-1 and dynein. These vesicles co-localize with markers for recycling endosomes and early but not late endosomes. They frequently undergo fission, providing a mechanism for the exclusion of ntcp from late endosomes. PI(3,4,5)P3 activates PKCzeta and enhances motility of the ntcp vesicles and overcomes the partial inhibition produced by a PI3-kinase inhibitor. Specific inhibition of PKCzeta blocks the motility of ntcp-containing vesicles but has no effect on late vesicles as shown both in vitro and in living cells transfected with ntcp-GFP. These data indicate that PKCzeta is required specifically for the intracellular movement of vesicles that contain the ntcp transporter.     

Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells

Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules.     

Platelet-activating factor acetylhydrolase (PAF-AH)

Platelet-activating factor (PAF) is one of the most potent lipid messengers involved in a variety of physiological events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity, and its deacetylation induces loss of activity. The deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH). A series of biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Type I PAF-AH is a G-protein-like complex consisting of two catalytic subunits (alpha1 and alpha2) and a regulatory beta subunit. The beta subunit is a product of the LIS1 gene, mutations of which cause type I lissencephaly. Recent studies indicate that LIS1/beta is important in cellular functions such as induction of nuclear movement and control of microtubule organization. Although substantial evidence is accumulating supporting the idea that the catalytic subunits are also involved in microtubule function, it is still unknown what role PAF plays in the process and whether PAF is an endogenous substrate of this enzyme. Type II PAF-AH is a single polypeptide and shows significant sequence homology with plasma PAF-AH. Type II PAF-AH is myristoylated at the N-terminus and like other N-myristoylated proteins is distributed in both the cytosol and membranes. Plasma PAF-AH is also a single polypeptide and exists in association with plasma lipoproteins. Type II PAF-AH as well as plasma PAF-AH may play a role as a scavenger of oxidized phospholipids which are thought to be involved in diverse pathological processes, including disorganization of membrane structure and PAF-like proinflammatory action. In this review, we will focus on the structures and possible biological functions of intracellular PAF-AHs.     

The interaction between newly-formed mRNA and ribosomal particles during retarded translation]

The formation of polyribosomes in mouse liver cells at the reduced-rate translation was studied by treatment with cycloheximide (CHI) and aurintricarboxylic (ATA) acid. An increase of polypeptide synthesis time by 1.7-2.7 times (0.5 mg CHI per 25 g of weight or 15 mg ATA per 25 g) leads to a delay of the entrance of newly formed cytoplasmic D-RNA into polyribosomes. These results are in agreement with the model of polyribosome formation from ribonucleoprotein precursors containing cytoplasmic D-RNA. On the other hand, in the presence of a CHI dose (5 mg/25 g) causing a dramatic (240-fold) increase of polypeptide synthesis time, the kinetics of entrance of newly formed D-RNA into polyribosomes does not differ from the normal one, and amount of the incorporated mRNA is even somewhat higher than under normal conditions. It is suggested that in this situation ribosomes are moving along the newly formed mRNA, and their movement is not accompanied by the synthesis of completed polypeptide chain.     

Pharmacological inhibition of the intracellular transport of low-density lipoprotein-derived cholesterol in Chinese hamster ovary cells

Mammalian cells, cultured in the presence of serum lipoproteins, acquire cholesterol necessary for growth from the uptake and lysosomal hydrolysis of low-density lipoproteins (LDL). The mechanism(s) of intracellular transport of LDL-derived cholesterol from lysosomes to other cellular sites is unknown. In this study, various pharmacological agents were assessed for their ability to inhibit the movement of LDL-cholesterol from lysosomes to the plasma membrane. The only pharmacological agent tested in these experiments that specifically inhibited LDL-cholesterol movement was U18666A. Ketoconazole impaired the intracellular transport of LDL-cholesterol; however, ketoconazole also had a general effect on cholesterol movement, since it impeded the desorption of endogenously synthesized cholesterol into the medium. Other drugs that affected cholesterol movement appeared to be nonspecific. Cholesterol transport from lysosomes to plasma membranes was not significantly altered by agents that affect lysosomal function or cytoskeletal organization, as well as energy poisons and cycloheximide.     

p38: A novel protein that associates with the vesicular stomatitis virus glycoprotein

The vesicular stomatitis virus glycoprotein (VSV G) is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. The cytoplasmic domain of VSV G contains information for several intracellular sorting steps including efficient export from the ER, basolateral delivery, and endocytosis. In order to identify proteins that potentially interact with the polypeptide sorting motifs in the VSV G tail, the carboxy-terminal 27 amino acids of VSV G were used as bait in a yeast two-hybrid system. The protein identified most frequently in the screen is a novel protein of 38 kDa, p38. In the present work, the initial molecular and biochemical characterization of p38 is described. Preliminary evidence suggests that p38 may interact transiently with endoplasmic reticulum (ER) membranes, and thus may affect VSV G and other cargo movement at the step of ER to Golgi traffic.     

The lysosomal polypeptide transporter TAPL: more than a housekeeping factor?

The transporter associated with antigen processing-like (TAPL) is a polypeptide transporter translocating cytosolic peptides into the lumen of lysosomes driven by ATP hydrolysis. TAPL belongs to the family of ABC transporters and forms a homodimer. This ABC transporter not only shows a broad tissue but also a wide phylogenetic distribution, because orthologs are still found in nematodes and insects. Here, we present the topology, substrate specificity, and distribution of this intracellular polypeptide transporter. Additionally, we will discuss its proposed physiological functions such as housekeeping together with a specialized factor for metabolite storage as well as for the adaptive immunity.     

Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis.

Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.     

Glucose deprivation induces the selective accumulation of hexose transporter protein GLUT-1 in the plasma membrane of normal rat kidney cells

Antibody to the carboxyl-terminal of hexose transporter protein GLUT-1 was used to localize this carrier in normal rat kidney (NRK) cells during D-glucose (Glc) deprivation. Glc-deprivation of NRK cells induces increased hexose transport, inhibits the glycosylation of GLUT-1, and increases the content of both native, 55,000 apparent mol wt (Mr) and aglyco, 38,000 Mr GLUT-1 polypeptides. The distribution of GLUT-1 protein in subcellular fractions isolated from Glc-fed NRK cells shows that the 55,000 Mr polypeptide is most abundant in intracellular membrane fractions. Glc-fed cells that have been tunicamycin treated contain principally the 38,000 Mr GLUT-1 polypeptide, which is found predominantly in intracellular membrane fractions. In Glc-deprived cells the 55,000 Mr GLUT-1 polypeptide localizes predominantly in the Golgi and plasma membrane fractions, whereas the more abundant 38,000 Mr GLUT-1 polypeptide is distributed throughout all membrane fractions. In Glc-deprived but fructose-fed cells only the 55,000 Mr GLUT-1 polypeptide is detected, and it is found predominantly in the plasma membrane and Golgi fractions. The localization of GLUT-1 protein was directly and specifically visualized in NRK cells by immunofluorescence microscopy. Glc-fed cells show little labeling of cell borders and a small punctate juxtanuclear pattern suggestive of localization to the Golgi and, perhaps, endoplasmic reticulum. Glc-fed cells that have been tunicamycin treated show large punctate intracellular accumulations suggestive of localization to distended Golgi and perhaps endoplasmic reticulum. Glc-deprived cells exhibited intense labeling of cell borders as well as intracellular accumulations. Glc-deprived but fructose-fed cells show fewer intracellular accumulations, and the labeling is, in general, limited to the cell borders. Our results suggest that Glc deprivation induces the selective accumulation of GLUT-1 in the plasma membrane of NRK cells.     

Cell-free synthesis of phytochrome apoprotein

A single polypeptide is immunospecifically precipitated by monospecific antiphytochrome from the total translation products of both wheat-germ and rabbit-reticulocyte cell-free protein synthesizing systems programmed with oat (Avena sativa L.) poly(A) RNA. The mobility of this polypeptide is slightly lower on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than that of immunoaffinity-purified, 118 kdalton phytochrome and corresponds to an apparent molecular weight of 124 kdalton. Evidence against the possibility that this mobility difference results from intracellular processing of the 124-kdalton protein is provided by extraction of freeze-dried tissue directly into boiling SDS-containing buffer. This procedure yields a phytochrome species with a mobility on SDS polyacrylamide gel electrophoresis indistinguishable from that of the in-vitro translation product. Together the data indicate that the phytochrome polypeptide is synthesized in its mature form in the cell but is subject to modification to a form with lower apparent molecular weight during immunopurification.Abbreviations IgG immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate