n higher eukaryotes, secretory and plasma-membrane proteins are transported from the endoplasmic reticulum (ER) to a central Golgi complex and subsequently packaged into membrane-bound carriers for delivery to the cell surface. The long-distance transport of post-Golgi organelles to the tips of axons or to developing hyphal extensions in Neurospora crassa shows an absolute requirement for microtubules and microtubule-associated motors 1,2 . In contrast, microtubule disruption only moderately attenuates Golgi-toplasma-membrane transport in fibroblasts and randomizes surface delivery of select proteins in epithelial cells 3,4 . The observed preservation of biosynthetic transport after microtubule disruption is probably due to the extensive fragmentation and redistribution of Golgi mini-stacks to regions immediately adjacent to both the ER and the plasma membrane 3,5,6 . Here we have designed experiments to test the hypothesis that when the characteristic central localization of the Golgi is preserved, microtubules, kinesin and the GTPase dynamin are essential for post-Golgi trafficking. We ruled out a pharmacological approach to tackling this problem because we found that microtubule antagonists caused dispersal of the Golgi complex before complete microtubule disassembly occurred (see Supplementary Information). Instead, we microinjected functionblocking anti-kinesin antibodies HD and SUK-4 or cDNAs encoding a dominant-negative form of dynamin into cells expressing a green fluorescent protein (GFP)-tagged apical-membrane protein, p75. We show that kinesin and dynamin are required for different stages of post-Golgi transport.During a 2.5-h transport block at 20 °C, newly synthesized p75-GFP translocated from the ER to a juxtanuclear region (Fig. 1a, control, 0 min) and co-localized with Golgi/trans-Golgi network (TGN) markers (Fig. 1b). Within 4 h after shifting to the permissive temperature for transport, 32 °C, 81% of p75-GFP translocated from the Golgi to the plasma membrane (Fig. 1a, control, 240 min). The emptying rate of p75-GFP from the Golgi correlated with its arrival at the cell surface, as determined by immunocytochemical analysis of p75 in injected cells (data not shown) and by pulsechase, surface-biotinylation assays of p75 or p75-GFP in stable MDCK transfectants (see Supplementary Information). Normal trafficking of p75 was unaffected by the GFP tag: microinjected p75-GFP was selectively delivered to the apical membrane of confluent, polarized MDCK cells (data not shown). Figure 1 Inhibition of kinesin activity or expression of D2K44E inhibits release of p75-GFP from the Golgi/TGN after release from a block at 20 °C. a, Cells were microinjected with p75-GFP alone (control; n = 19) or with p75-GFP and HD (n = 13), SUK-4 (n = 8), wild-type dynamin-2 cDNA (n = 9) or cDNA encoding a dominantnegative dynamin-2, D2K44E (n = 15). Exit of p75-GFP from the Golgi/TGN was monitored by time-lapse imaging after shifting to 32 °C. First (0 min) and last (240 min) images from representative recordings are shown. In ...

The GTPase dynamin has been implicated in the regulation of the scission of coated and noncoated pits during the early stages of endocytosis. Various macromolecules including microtubules, acidic phospholipids, and Src homology 3 (SH3) domains have been shown to interact with the basic, proline-rich region of dynamin and act as effectors of its GTPase activity. The interaction of dynamin with SH3 domain-containing proteins is of particular interest since SH3 domains are known to mediate protein-protein interactions in signal transducing complexes. In this study, we have systematically defined three distinct SH3 binding regions within the dynamin proline-rich C terminus. These binding regions conform to either the Class I or II SH3 binding consensus sequence, and their location coincides with a region previously shown to be important in the colocalization of dynamin with clathrin-coated pits. Two of these SH3 binding regions are well conserved among four dynamin isoforms, and we show that the overall binding pattern for SH3 domains is comparable among the isoforms. We also demonstrate that neither transferrin nor plateletderived growth factor receptor uptake is restored upon removal of the basic, proline-rich region in a dominant negative dynamin GTP binding mutant. Together with earlier evidence from our laboratory, these findings suggest that SH3 domains may serve to target dynamin to coated pits and are not the direct targets of dominant inhibitory mutants of dynamin.

Dynamin, a 100-kDa GTPase, has been implicated to be involved in synaptic vesicle recycling, receptor-mediated endocytosis, and other membrane sorting processes. Dynamin self-assembles into helical collars around the necks of coated pits and other membrane invaginations and mediates membrane scission. In vitro, dynamin has been reported to exist as dimers, tetramers, ring-shaped oligomers, and helical polymers. In this study we sought to define self-assembly regions in dynamin. Deletion of two closely spaced sequences near the dynamin-1 C terminus abolished self-association as assayed by co-immunoprecipitation and the yeast interaction trap, and reduced the sedimentation coefficient from 7.5 to 4.5 S. Circular dichroism spectroscopy and equilibrium ultracentrifugation of synthetic peptides revealed coiled-coil formation within the C-terminal assembly domain and at a third, centrally located site. Two of the peptides formed tetramers, supporting a role for each in the monomer-tetramer transition and providing novel insight into the organization of the tetramer. Partial deletions of the C-terminal assembly domain reversed the dominant inhibition of endocytosis by dynamin-1 GTPase mutants. Self-association was also observed between different dynamin isoforms. Taken altogether, our results reveal two distinct coiled-coilcontaining assembly domains that can recognize other dynamin isoforms and mediate endocytic inhibition. In addition, our data strongly suggests a parallel model for dynamin subunit self-association.

Dynamin is a GTPase involved in endocytosis and other aspects of membrane trafficking. A critical function in the presynaptic compartment attributed to the brain-specific dynamin isoform, dynamin-1, is in synaptic vesicle recycling. We report that dynamin-2 specifically interacts with members of the Shank/ProSAP family of postsynaptic density scaffolding proteins and present evidence that dynamin-2 is specifically associated with the postsynaptic density. These data are consistent with a role for this otherwise broadly distributed form of dynamin in glutamate receptor down-regulation and other aspects of postsynaptic membrane turnover.Dynamin is a 100-kDa GTPase (1, 2) that controls a variety of vesicular budding events including synaptic vesicle recycling, receptor-mediated endocytosis, caveolae internalization, phagocytosis, and secretory vesicle budding from the transGolgi network (3-9). It forms long spiral polymers around the necks of coated pits (10) and on lipid tubules (11), suggesting that the protein may directly function in membrane scission. Alternatively, dynamin has also been postulated to act as a GTPase switch by recruiting other endocytic factors to the neck and then activating them to sever the coated vesicle (12).Dynamin contains an amino-terminal GTPase domain, followed by a central coiled-coil assembly domain (13), a pleckstrin homology domain, which binds to phosphoinositides and the ␤␥ subunits of heterotrimeric GTPases (14,15), and a carboxyl-terminal coiled-coil region (also called the assembly or GTPase effector domain) that is involved in self-association (13,16,17). At the extreme carboxyl terminus is a basic, prolinerich domain to which a number of Src homology 3 (SH3) 1 domain-containing proteins, acidic phospholipids, and microtubules have been shown to bind (18 -20).Considerable insight into dynamin function at the synapse has come from genetic and morphological studies on the temperature-sensitive mutants of shibire, the dynamin ortholog in Drosophila (21,22). Single point mutations in the GTPase domain of shibire cause paralysis at elevated temperatures, and ultrastructural analysis of nerve terminals under these conditions has revealed a depletion of synaptic vesicles, along with an accumulation of collared pits (23,24).In mammals, three closely related dynamin genes are expressed in a tissue-specific manner. Dynamin-1 is almost exclusively expressed in neurons (25). Dynamin-2 is found in the brain but is also widely expressed among other tissues (26 -28). Dynamin-3 was initially identified in testis (29) but is also found in brain, lung, and heart (30). Differences in the subcellular distribution of the dynamin gene products and their alternative splice forms have been reported (30). Because of its restriction to neurons, dynamin-1 has been assumed to be the synaptic isoform. The function of dynamin-2 is less well understood, and a role in neurons has not been identified. Overexpression of a dominant inhibitory mutant form of dynamin-2 in cultured hippocampal neurons was recen...

The nit-3 gene of the filamentous fungus Neurospora crassa encodes the enzyme nitrate reductase, which catalyzes the first reductive step in the highly regulated nitrate assimilatory pathway. The nucleotide sequence of nit-3 was determined and translates to a protein of 982 amino acid residues with a molecular weight of approximately 108 kDa. Comparison of the deduced nit-3 protein sequence with the nitrate reductase protein sequences of other fungi and higher plants revealed that a significant amount of homology exists, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The synthesis and turnover of the nit-3 mRNA were also examined and found to occur rapidly and efficiently under changing metabolic conditions.