Direct binding of cytosolic NDP kinases to membrane lipids is regulated by nucleotides.

In spite of their complete lack of any structural features that characterize membrane proteins, cytosolic nucleoside-diphosphate kinases (NDPKs) have been found repeatedly to associate with membranes. In some instances the recruitment of cytosolic NDPKs to membranes was attributed to interactions with peripheral or integral membrane proteins, but in many cases the mechanism underlying the association of NDPKs with membranes remained unknown. We show here that cytosolic NDPKs bind directly to membrane lipids in a dynamic process that is controlled by its substrates, nucleoside tri- and diphosphates, and can be fully reconstituted with chemically defined, protein-free phospholipids and recombinant NDPK, or with purified NDPK.

Methods for the isolation of sensory and primary cilia--an overview.

Detailed proteomic analyses of mammalian olfactory and rod photoreceptor sensory cilia are now available, providing an inventory of resident ciliary proteins and laying the foundation for future studies of developmental and spatiotemporal changes in the composition of sensory cilia. Cilia purification methods that were elaborated and perfected over several decades were essential for these advances. In contrast, the proteome of primary cilia is yet to be established, because purification procedures for this organelle have been developed only recently.

NDP kinase moves into developing primary cilia.

Inmunofluorescence staining of murine NIH3T3 fibroblasts grown at high density shows that conventional nucleoside diphosphate (NDP) kinases A and B localize to a sensory organelle, the primary cilium. Similar results are obtained with Xenopus A6 kidney epithelial cells, suggesting that NDP kinases are a universal component of the primary cilium. The translocation of NDP kinase into primary cilia depends on size, taking place only when cilia reach a critical length of 5-6 microm.

Receptor activation regulates cortical, but not vesicular localization of NDP kinase.

We used immunofluorescence techniques to determine the localization of nucleoside diphosphate (NDP) kinase in NIH-3T3 fibroblasts. We found that cytoplasmic NDP kinase can be separated into two populations according to subcellular localization and response to extracellular stimuli. Specifically, within minutes of stimulation of resting fibroblasts with serum, growth factors or bombesin, a portion of NDP kinase becomes associated with membrane ruffles and lamellipodia.