A novel motor, KIF13A, transports mannose-6-phosphate receptor to plasma membrane through direct interaction with AP-1 complex.

Intracellular transport mediated by kinesin superfamily proteins (KIFs) is a highly regulated process. The molecular mechanism of KIFs binding to their respective cargoes remains unclear. We report that KIF13A is a novel plus end-directed microtubule-dependent motor protein and associates with beta 1-adaptin, a subunit of the AP-1 adaptor complex. The cargo vesicles of KIF13A contained AP-1 and mannnose-6-phosphate receptor (M6PR). Overexpression of KIF13A resulted in mislocalization of the AP-1 and the M6PR. Functional blockade of KIF13A reduced cell surface expression of the M6PR. Thus, KIF13A transports M6PR-containing vesicles and targets the M6PR from TGN to the plasma membrane via direct interaction with the AP-1 adaptor complex.

Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport.

Experiments with vesicles containing N-methyl-D-aspartate (NMDA) receptor 2B (NR2B subunit) show that they are transported along microtubules by KIF17, a neuron-specific molecular motor in neuronal dendrites. Selective transport is accomplished by direct interaction of the KIF17 tail with a PDZ domain of mLin-10 (Mint1/X11), which is a constituent of a large protein complex including mLin-2 (CASK), mLin-7 (MALS/Velis), and the NR2B subunit. This interaction, specific for a neurotransmitter receptor critically important for plasticity in the postsynaptic terminal, may be a regulatory point for synaptic plasticity and neuronal morphogenesis.

Kinesin superfamily protein 3 (KIF3) motor transports fodrin-associating vesicles important for neurite building.

Kinesin superfamily proteins (KIFs) comprise several dozen molecular motor proteins. The KIF3 heterotrimer complex is one of the most abundantly and ubiquitously expressed KIFs in mammalian cells. To unveil the functions of KIF3, microinjection of function-blocking monovalent antibodies against KIF3 into cultured superior cervical ganglion (SCG) neurons was carried out. They significantly blocked fast axonal transport and brought about inhibition of neurite extension. A yeast two-hybrid binding assay revealed the association of fodrin with the KIF3 motor through KAP3. This was further confirmed by using vesicles collected from large bundles of axons (cauda equina), from which membranous vesicles could be prepared in pure preparations. Both immunoprecipitation and immunoelectron microscopy indicated the colocalization of fodrin and KIF3 on the same vesicles, the results reinforcing the evidence that the cargo of the KIF3 motor consists of fodrin-associating vesicles. In addition, pulse-labeling study implied partial comigration of both molecules as fast flow components. Taken together, the KIF3 motor is engaged in fast axonal transport that conveys membranous components important for neurite extension.

Uso1 protein is a dimer with two globular heads and a long coiled-coil tail.

USO1 is one of the essential genes in Saccharomyces cerevisiae whose gene products participate in protein transport from the endoplasmic reticulum to the Golgi apparatus. This product was purified to homogeneity. Electron microscopic study revealed that it has a single or double globular domain with a long tail and that the molecule is a dimer. A peak position of the distribution of rod length was 154.5 nm, in agreement with the secondary structure prediction that it has a long alpha-helix at the carboxyl terminus. Probability of coiled-coil formation was also predicted from the primary structure of the product, which asserts that it has a long alpha-helical coiled-coil at the carboxyl-terminal region with some interruptions. Certainly, the electron microscopic image of this molecule had some hinges within the rod region. The distance was measured between the globular domain and the hinges. Two peaks of the distribution of the hinge position exist at 23.1 and 85.5 nm from the globular domain. This is consistent with the predicted positions of interruption. These results give new experimental evidence that Uso1 protein is a dimer and has an alpha-helical coiled-coil tail with two globular heads.

Calcium and SLY genes suppress the temperature-sensitive secretion defect of Saccharomyces cerevisiae uso1 mutant.

Saccharomyces cerevisiae uso1-1 mutant stops the transport of secretory proteins from the endoplasmic reticulum to the Golgi apparatus at 37 degrees C. We found that this temperature-sensitive defect was suppressed either by increasing the concentration of calcium ion in the medium or by introducing in the cell the SLY genes which suppress the defect of Ypt1 protein, a small GTP-binding protein. The common phenotype and suppression of the mutants suggest that Uso1 and Ypt1 proteins function in the same process of protein transport, i.e., targeting or fusion of the transport vesicles to the Golgi membrane.

Uso1 protein contains a coiled-coil rod region essential for protein transport from the ER to the Golgi apparatus in Saccharomyces cerevisiae.

We have previously shown that the Saccharomyces cerevisiae USO1 gene required in the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus encodes a 200-kDa protein (1,790 amino acids) which is present in a nonglobular high molecular mass complex. Antibodies against an N-terminal portion of Uso1 protein recognized a 100-kDa protein in Western blot of the temperature-sensitive uso1-1 mutant cell lysate. The nucleotide sequence of uso1-1 indicated the 951st codon was UAG (amber) in place of CAG (glutamine) in USO1. Deletion study of USO1 gene indicated that such truncated Uso1 polypeptides are sufficiently functional at 25 degrees C but not at 37 degrees C. Mutant Uso1-1 protein displayed an apparent molecular mass of 400-500 kDa in gel filtration while it cosedimented with a globular 6S marker protein, horseradish peroxidase (44 kDa), in sucrose density gradient centrifugation. These results indicated that truncated Uso1-1 protein is still present in a nonglobular high molecular mass complex, similar to the wild-type Uso1 protein.

Molecular characterization of the USO1 gene product which is essential for vesicular transport in Saccharomyces cerevisiae.

We have previously shown that USO1 gene required in the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus encodes a hydrophilic protein of 1790 amino acids. The sequence of carboxyl-terminal 1010 amino acids was predicted to have an alpha-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins. Antibodies raised against partial sequences of the Uso1 polypeptide reacted with a 200 kDa protein in Western blots of the wild-type yeast proteins. The Uso1 protein was found predominantly in the soluble fraction and displayed a molecular mass of 800-900 kDa in gel filtration when globular protein were used as molecular mass standards. In sucrose density gradient centrifugation, however, the Uso1 protein cosedimented with a globular 6S marker protein, horseradish peroxidase (44 kDa). These results suggest that, in its native state, the Uso1 protein forms a nonglobular oligomer.

SS33410, an inhibitor for inflammation, blocks the intracellular transport of VSV G glycoprotein in BHK cells.

We have studied the effect of SS33410, an inhibitor for inflammation, on the intracellular transport and processing of the vesicular stomatitis virus (VSV) G glycoprotein as a model integral membrane protein. Delivery of G glycoprotein to the cell surface was blocked by 0.5 microgram/ml of SS33410 without any significant inhibition of protein synthesis. The G glycoprotein accumulated intracellularly electrophoresed a little faster than the control mature one excreted to the medium. The affinity for concanavalin A-agarose (Con-A) column suggested that most of the G glycoprotein oligosaccharides were of the high-mannose type. These results indicate that processing of N-glycosidic oligosaccharide is incomplete, suggesting that intracellular trafficking is arrested before reaching to the trans Golgi compartments in the presence of SS33410.