Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes.

Localization of bicoid (bcd) mRNA to the anterior and oskar (osk) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. Previous genetic studies implicated exuperantia (exu) in bcd mRNA localization, but its role in this process is not understood. We have biochemically isolated Exu and show that it is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins was identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which we show binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the Exu-Yps complex contains osk mRNA. This biochemical result led us to reexamine the role of Exu in the localization of osk mRNA. We discovered that exu-null mutants are defective in osk mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and osk mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. We propose that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte.

Single-molecule behavior of monomeric and heteromeric kinesins.

Conventional kinesin is capable of long-range, processive movement along microtubules, a property that has been assumed to be important for its role in membrane transport. Here we have investigated whether the Caenorhabditis elegans monomeric kinesin unc104 and the sea urchin heteromeric kinesin KRP85/95, two other members of the kinesin superfamily that function in membrane transport, are also processive. Both motors were fused to green fluorescent protein, and the fusion proteins were tested for processive ability using a single-molecule fluorescence imaging microscope. Neither unc104-GFP nor KRP85/95-GFP exhibited processive movement (detection limit approximately 40 nm), although both motors were functional in multiple motor microtubule gliding assays (v = 1760 +/- 540 and 202 +/- 37 nm/s, respectively). Moreover, the ATP turnover rates (5.5 and 3.1 ATPs per motor domain per second, respectively) are too low to give rise to the observed microtubule gliding velocities, if only a single motor were driving transport with an 8 nm step per ATPase cycle. Instead, the results suggest that these motors have low duty cycles and that high processivity may not be required for efficient vesicle transport. Conventional kinesin's unusual processivity may be required for efficient transport of protein complexes that cannot carry multiple motors.

The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain.

Members of the kinesin superfamily share a similar motor catalytic domain yet move either toward the plus end (e.g., conventional kinesin) or the minus end (e.g., Ncd) of microtubules. The structural features that determine the polarity of movement have remained enigmatic. Here, we show that kinesin's catalytic domain (316 residues) in a dimeric construct (560 residues) can be replaced with the catalytic domain of Ncd and that the resultant motor moves in the kinesin direction. We also demonstrate that this chimera does not move processively over many tubulin subunits, which is similar to Ncd but differs from the highly processive motion of conventional kinesin. These findings reveal that the catalytic domain contributes to motor processivity but does not control the polarity of movement. We propose that a region adjacent to the catalytic domain serves as a mechanical transducer that determines directionality.

Microtubule interaction site of the kinesin motor.

Kinesin and myosin are motor proteins that share a common structural core and bind to microtubules and actin filaments, respectively. While the actomyosin interface has been well studied, the location of the microtubule-binding site on kinesin has not been identified. Using alanine-scanning mutagenesis, we have found that microtubule-interacting kinesin residues are located in three loops that cluster in a patch on the motor surface. The critical residues are primarily positively charged, which is consistent with a primarily electrostatic interaction with the negatively charged tubulin molecule. The core of the microtubule-binding interface resides in a highly conserved loop and helix (L12/alpha5) that corresponds topologically to the major actin-binding domain of myosin. Thus, kinesin and myosin have developed distinct polymer-binding domains in a similar region with respect to their common catalytic cores.

Cloning and localization of a conventional kinesin motor expressed exclusively in neurons.

Kinesin is a microtubule-based motor protein involved in organelle transport in neuronal and nonneuronal cells. Although a single kinesin motor has been thought to serve all cell types, we document here that neurons express a second conventional kinesin heavy chain (nKHC) that is 65% identical in amino acid sequence to the ubiquitously expressed kinesin heavy chain (uKHC). By preparing antibodies which distinguish between the two KHCs, we demonstrate that nKHC is a nucleotide-dependent microtubule-binding protein which partially cofractionates with membrane organelles. Immunolocalization experiments show that nKHC is distributed throughout the CNS but is highly enriched in subsets of neurons. In hippocampal neurons in culture, uKHC is distributed uniformly throughout the neuron, whereas nKHC is selectively concentrated in the cell body. These results demonstrate that mammalian neuronal tissue contains two conventional kinesin motors which may serve distinct functions in microtubule-based transport.

Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells.

To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances.