Dynein motility: four heads are better than two

Cytoplasmic dynein is a microtubule-based motor protein that transports membranes in cells. The movement driven by a single dynein molecule in vitro is not as robust as dynein-driven movements in cells. A new study suggests that transport by multiple dyneins is more similar to cellular motions.     

The NADPH oxidase components p47(phox) and p40(phox) bind to moesin through their PX domain.

The NADPH oxidase of phagocytes is a membrane-bound heterodimeric flavocytochrome which catalyses the transfer of electrons from NADPH in the cytoplasm to oxygen in the phagosome. A number of cytosolic proteins are involved in its activation/deactivation: p47phox, p67phox, p40phox and the small GTP-binding protein, rac. The cytosolic phox proteins interact with the cytoskeleton in human neutrophils and, in particular, an interaction with coronin has been reported (Grogan A., Reeves, E., Keep, N. H., Wientjes, F., Totty, N., Burlingame, N. L., Hsuan, J., and Segal, A. W. (1997) J. Cell Sci. 110, 3071-3081). Here, we report on the interaction of another cytoskeletal protein, moesin, with the phox proteins. Moesin belongs to the ezrin-radixin-moesin family of F-actin-binding proteins and we show that it binds to p47phox and p40phox in a phosphoinositide-dependent manner. Furthermore, we show that its N-terminal part binds to the PX domain of p47phox and p40phox.     

Pharmacological chaperoning: two 'hits' are better than one

Protein folding disorders comprise a rapidly growing group of diseases that involve virtually every organ system and affect individuals of all ages. Their principal pathology is the inability of a protein to acquire or maintain its physiological three-dimensional structure. In cells, this generally results in one of three outcomes: accumulation of misfolded protein aggregates, cell death, or recognition by cellular quality control machinery and rapid degradation. Large-scale screening efforts to identify and design small molecules that either repair the folding defect or enable the protein to escape degradation have been encouraging. However, most compounds identified to date restore only a small fraction of molecules to the normal folding pathway, and hence are relatively poor therapeutic candidates. Results published by Wang et al. in this issue of the Biochemical Journal show that, for mutant forms of two ABC (ATP-Binding-Cassette) transporters, P-glycoprotein and CFTR (cystic fibrosis transmembrane conductance regulator), modest correction of trafficking by single agents can be additive when multiple compounds are used in combination. These findings raise the intriguing possibility that corrector molecules acting at different steps along the folding pathway might provide a multidrug approach to human protein folding disorders.     

Bird communication: two voices are better than one

How do emperor penguins find their mates on a featureless ice flow, packed at densities of ten animals per square meter? A recent study has revealed how use of their 'two-voice' calls enables emperor penguins to locate their mates and chicks under some of nature's most extreme conditions.     

Protein transport: two translocons are better than one

The translocon is the gateway to the endoplasmic reticulum (ER). In yeast this is the Sec61p complex. However, new evidence suggests that a second translocon containing the Sec61p homolog Ssh1p provides important flexibility to the ER translocation machinery.     

Circadian clockwork: two loops are better than one

The spectacularly successful race over the past three years to place our understanding of the circadian clockwork of mammals into a molecular framework is beginning to yield the cardinal example of the molecular-genetic control of behaviour. This perspective describes recent evidence for the conservation of a double-loop, autoregulatory feedback mechanism across the best understood eukaryotic circadian systems, and discusses how these findings may illuminate some long-standing puzzles concerning our subliminal sense of circadian time.     

Membrane binding mechanisms of the PX domains of NADPH oxidase p40phox and p47phox

Phox (PX) domains are phosphoinositide (PI)-binding domains with broad PI specificity. Two cytosolic components of NADPH oxidase, p40(phox) and p47(phox), contain PX domains. The PX domain of p40(phox) specifically binds phosphatidylinositol 3-phosphate, whereas the PX domain of p47(phox) has two lipid binding sites, one specific for phosphatidylinositol 3,4-bisphosphate and the other with affinity for phosphatidic acid or phosphatidylserine. To delineate the mechanisms by which these PX domains interact with PI-containing membranes, we measured the membrane binding of these domains and respective mutants by surface plasmon resonance and monolayer techniques and also calculated the electrostatic potentials of the domains as a function of PI binding. Results indicate that membrane binding of both PX domains is initiated by nonspecific electrostatic interactions, which is followed by the membrane penetration of hydrophobic residues. The membrane penetration of the p40(phox) PX domain is induced by phosphatidylinositol 3-phosphate, whereas that of the p47(phox) PX domain is triggered by both phosphatidylinositol 3,4-bisphosphate and phosphatidic acid (or phosphatidylserine). Studies of enhanced green fluorescent protein-fused PX domains in HEK293 cells indicate that this specific membrane penetration is also important for subcellular localization of the two PX domains. Further studies on the full-length p40(phox) and p47(phox) proteins showed that an intramolecular interaction between the C-terminal Src homology 3 domain and the PX domain prevents the nonspecific monolayer penetration of p47(phox), whereas such an interaction is absent in p40(phox).     

Cargo transport: two motors are sometimes better than one

Molecular motor proteins are crucial for the proper distribution of organelles and vesicles in cells. Much of our current understanding of how motors function stems from studies of single motors moving cargos in vitro. More recently, however, there has been mounting evidence that the cooperation of multiple motors in moving cargos and the regulation of motor-filament affinity could be key mechanisms that cells utilize to regulate cargo transport. Here, we review these recent advances and present a picture of how the different mechanisms of regulating the number of motors moving a cargo could facilitate cellular functions.     

Odor recognition memory: two encoding trials are better than one

The primary purpose of this study was to determine the effect of one versus two encoding trials in the classical yes/no recognition memory paradigm using olfactory stimuli. A group of 24 young adults rated 18 standard microencapsulated odorant targets for familiarity (first encoding block) or pleasantness (second encoding block). Once-encoded targets were in only one block and twice-encoded targets were in both, with items counterbalanced across participants. Participants performed a 20-min nonverbal distractor task followed by a yes/no recognition test incorporating 18 additional odors as foils. Memory performance for twice-encoded targets was superior to that for once-encoded targets. For once-encoded targets, performance did not differ between those rated for familiarity versus those rated for pleasantness. Less pleasant odors produced overall better recognition, with a tendency for less familiar odors to produce overall better recognition. There was a tendency for the second encoding trial to have a larger effect for less pleasant or familiar odors than for more pleasant or familiar odors. The main conclusion is that recognition memory for odors is better for items encoded two times than for items encoded only once. Implications of these findings and suggestions for future research are discussed.     

Antifreeze protein dimer: when two ice-binding faces are better than one

A naturally occurring tandem duplication of the 7-kDa type III antifreeze protein from Antarctic eel pout (Lycodichthys dearborni) is twice as active as the monomer in depressing the freezing point of a solution. We have investigated the basis for this enhanced activity by producing recombinant analogues of the linked dimer that assess the effects of protein size and the number and area of the ice-binding site(s). The recombinant dimer connected by a peptide linker had twice the activity of the monomer. When one of the two ice-binding sites was inactivated by site-directed mutagenesis, the linked dimer was only 1.2 times more effective than the monomer. When the two monomers were linked through a C-terminal disulfide bond in such a way that their two ice-binding sites were opposite each other and unable to engage the same ice surface simultaneously, the dimer was again only 1.2 times as active as the monomer. We conclude from these analyses that the enhanced activity of the dimer stems from the two ice-binding sites being able to engage to ice at the same time, effectively doubling the area of the ice-binding site.     

p47(phox) PX domain of NADPH oxidase targets cell membrane via moesin-mediated association with the actin cytoskeleton

Activation of phagocytic NADPH oxidase requires association of its cytosolic subunits with the membrane-bound flavocytochrome. Extensive phosphorylation of the p47(phox) subunit of NADPH oxidase marks the initiation of this activation process. The p47(phox) subunit then translocates to the plasma membrane, bringing the p67(phox) subunit to cytochrome b558 to form the active NADPH oxidase complex. However, the detailed mechanism for targeting the p47(phox) subunit to the cell membrane during activation still remains unclear. Here, we show that the p47(phox) PX domain is responsible for translocating the p47(phox) subunit to the plasma membrane for subsequent activation of NADPH oxidase. We also demonstrate that translocation of the p47(phox) PX domain to the plasma membrane is not due to interactions with phospholipids but rather to association with the actin cytoskeleton. This association is mediated by direct interaction between the p47(phox) PX domain and moesin.     

The p40phox and p47phox PX domains of NADPH oxidase target cell membranes via direct and indirect recruitment by phosphoinositides.

The Phox homology (PX) domain has recently been reported to bind to phosphoinositides, and some PX domains can localize to endosomes in vivo. Here we show data to support the conclusion that the p40(phox) PX domain binds to phosphatidylinositol 3-phosphate specifically in vitro and localizes to endosomes in intact cells. In addition, its Y59A/L65Q mutant, which has decreased affinity for phosphatidylinositol 3-phosphate in vitro, fails to target EGFP-p40-PX to endosomes. However, unlike published results, we find that the p47(phox) PX domain weakly binds to many phosphoinositides in vitro showing slightly higher affinity for phosphatidylinositol 3,4,5-trisphosphate. Moreover, we show for the first time that upon insulin-like growth factor-1 stimulation of COS cells, the p47(phox) PX domain is localized to the plasma membrane, and this subcellular localization is dependent on PI 3-kinase activity. Unexpectedly, its R42Q mutant that loses in vitro phosphoinositide-binding ability can still target EGFP-p47-PX to the plasma membrane. Our data suggest that the translocation of p47(phox) PX domain to the plasma membrane does involve 3'-phosphoinositide(s) in the process, but the phosphoinositide-binding of p47(phox) PX domain is not sufficient to recruit it to the plasma membrane. Therefore, the p40(phox) and p47(phox) PX domains can target subcellular membranes via direct or indirect recruitment by phosphoinositides, while both are under the control of phosphatidylinositol 3-kinase activity.