Going uninhibited with tail-less motors

HEADLINES

There is more than just sugar in the recipe for jelly An egg goes through a series of mysterious processes as it readies for encountering sperm. In frogs, as the egg passes through the oviduct, it is coated with a series of layers – rich glycoconjugates and proteins – that comprise the jelly coat of the egg. The structure and importance of the egg jelly has long been known – in Xenopus, it comprises three distinct layers, and removal of the jelly renders the egg unfertilizable. Recently, two laboratories have begun characterizing the jelly factors that are required for fertilization. As a first step towards identifying the jelly molecules involved in fertilization, Mozingo and Hedrick1 provided a detailed description of the distribution of the sugar moieties comprising much of the structural components of Xenopus egg jelly. The differences in

sugar distribution and abundance in each jelly sublayer was determined by lectin binding. Preincubating eggs with some lectins inhibited sperm penetration of the jelly, suggesting that the sugar moieties might be involved in sperm binding. By contrast, Olson and Chandler2 report that proteins that can diffuse from the jelly are both necessary and sufficient for restoring sperm binding to jellyless eggs. Significantly, neither the structural components nor a range of charged polymers could restore fertilization to jellyless eggs. The diffusible factor must be present at the time of fertilization as pretreating jellyless eggs with the diffusible fraction and then removing it prior to sperm addition did not promote fertilization. The fertilization-restoration activity is probably a protein as it is destroyed by heat and

protease treatment and is small – 60% of the activity is able to pass through a filter possessing a 50 kDa cut off. Although candidates for this diffusible factor have not been reported, once identified, it will be important to understand the relationships between the protein and glycoconjugate ingredients of the egg jelly in order to reconcile the interesting differences between these two studies.

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Mozingo, N. M. and Hedrick, J. L. (1999) Distribution of lectin binding sites in Xenopus laevis egg jelly, Dev. Biol. 210, 428–439 Olson, J. H. and Chandler, D. E. (1999) Xenopus laevis egg jelly contains small proteins that are essential to fertilization, Dev. Biol. 210, 401–410

Getting into thylakoids A dynamic synergy currently holds sway in the study of protein export across the bacterial inner membrane and chloroplast thylakoids. Initially, ideas flowed from the eubacterial to the thylakoid field, with chloroplast workers confirming the presence of Sec, signalrecognition particle (SRP) and spontaneous pathways in their system. Then, analysis of the maize mutant hcf106 led to the discovery of a novel export pathway where the trans-thylakoidal pH gradient drives certain folded proteins from the chloroplast stroma across the thylakoid membrane. A comparable DpH pathway was soon demonstrated in Escherichia coli, complete with Hcf106 homologues (tat A and tat E) and a characteristic twin-arginine motif in the N-terminal signal sequences of export substrates. Now, reports from two

This month’s headlines were contributed by Søren Andersen, Donald Gullberg, Robin May, Michael Mishkind and Kirsten Sadler.

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chloroplast labs1,2 suggest that thylakoids contain several distinct translocation machineries through which exported proteins transverse the membrane. The Hoffman and Cline groups find that antibodies to cpSecY (the chloroplast homologue of an E. coli translocation channel protein) inhibit translocation of Sec pathway, but not DpH pathway, substrates. Thus, thylakoids (and E. coli inner membranes as well) probably export Sec and DpH pathway substrates through different channels. In the case of the Sec and SRP pathways, however, data from E. coli point to a convergence at the level of the SecYEG/Sec61 translocation channel. Thus, it was surprising that Cline’s group could not inhibit translocation of a substrate for the SRP pathway with

the antibodies to cpSecY. Are all SRP pathway substrates in chloroplasts SecY independent, and, if so, what is the nature of the translocation machinery employed by these proteins? Clearly, much that is of interest continues to flow from the thylakoid system.

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Schuenemmann, D., Amin, P., Hartmann, E. and Hoffman, N. E. (1999) Chloroplast SecY is complexed to SecE and involved in the translocation of the 33-kDa but not the 23-kDa subunit of the oxygen-evolving complex, J. Biol. Chem. 274, 12177–12182 Mori, H., Summer, E. J., Ma, X. and Cline, K. (1999) Component specificity for the thylakoidal Sec and DpHdependent protein transport pathways, J. Cell Biol. 146, 45–55

Going uninhibited with tail-less motors Microtubule-based motor proteins read the polarity of microtubules (MTs) and spatially sort cargo in the cell. What do motors do when no cargo is bound? If they could move without cargo, one could imagine motors accumulating at MT ends, which would result in subse-

quent logistic problems combined with significant ATP/energy consumption. It seems reasonable to consider that the abundant pool of motors without cargo are inhibited somehow; previous biochemical and structural studies suggested that the tail of the MT-based

motor kinesin binds to cargo and also serves to inhibit non-cargo-bound motors. Two mutually complementing papers from the Howard1 and Vale2 laboratories show that the tail inhibits cargofree kinesin motility. The success of

trends in CELL BIOLOGY (Vol. 9) November 1999

headlines these experiments1,2 required performance of experiments in solution; previous motility experiments most likely failed to establish the inhibitory role of the tail convincingly because binding of full-length motor to a (glass) surface mimics cargo binding2. Experiments in solution showed that deletion of the tail increased the kinesin ATPase activity1,2 and, likewise, binding to cargo (glass beads)1; addition of a 64-amino-acid tail domain caused ATPase inhibition in both cases. Moreover, the tail domain completely and non-competitively

inhibited motility of the tail-less kinesin in an MT gliding assay1. Observations of the movement of single kinesin molecules on glassattached MTs (axonemes) revealed that the most significant difference between motors with and without a tail was a tenfold lower frequency of motor engagement and movement of the full-length motor; when moving, the full-length and tail-less motors moved equally fast2. It is suggested that the tail somehow (nonsterically1) inhibits both the ATPase

activity and the MT-binding ability of kinesin and that cargo binding alleviates these inhibitions1,2.

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Coy, D. L., Hancock, W. O., Wagenbach, M. and Howard, J. (1999) Kinesin’s tail domain is an inhibitory regulator of the motor domain, Nat. Cell Biol. 1, 288–292 Friedman, D. S. and Vale, R. D. (1999) Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain, Nat. Cell Biol. 1, 293–297

Mice without bite In recent years, genetic evidence from human diseases has highlighted the importance of cell adhesion for the integrity of skin and muscle. In the skin-blistering disease epidermolysis bullosa, genetic defects in keratins, integrin a6b4 or laminin-5 (a3b3g2) underlie the observed defects. Now, additional evidence for the importance of the individual molecules involved in cell adhesion comes from gene-knockout experiments in mice. The laminin a3 chain exists as two variants – a full-length and a truncated form – that are present in a number of laminin isoforms and show a wide expression pattern in different epithelia. In the paper by Ryan et al.1, the laminin a3 chain was inactivated in mice. The absence of all these variants causes a number of defects,

which lead to neonatal death. Three major defects were further analysed. First, in skin, blistering is observed and integrin a6b4 shows a patchy distribution. Surprisingly, integrin a3b1 is still able to bind to the laminin-5-defective basement membrane, indicating the presence of a hitherto unknown a3b1 ligand. Based on data from other systems it is speculated that this ligand is laminin-10. Second, epithelial cells defective in synthesis of the laminin a3 chain in vitro failed to survive on an unplated culture dish. This defect could be rescued if exogenous laminin-5 was added or, alternatively, when ligands for b1 or b4 integrins were supplied. Finally, a developmental defect in incisors arising from defective amelo-

blast differentiation was observed in mutant animals. In human junctional epidermolysis bullosa caused by defects in laminin-5, the phenotype includes skin blistering, dental abnormalities and high morbidity. Further analysis of the LAMA3-knockout mice should increase the understanding of the role the laminin a3 chain plays in tissue organization, gene expression and epithelial cell survival.

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Ryan, M. C., Lee, K., Miyashita, Y. and Carter, W. G. (1999) Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells, J. Cell Biol. 145, 1309–1323

Going local: rethinking Rho behaviour at the membrane Radical changes in biology are often so subtle that they pass unnoticed. Cell signalling is one area that has been in need of a concept change for some time – until recently, we all believed that our favourite signalling protein was multifunctional, and that rival candidates were mere imposters! Slowly, though, realization is dawning that the key to understanding signalling lies with localization. Protein X might phosphorylate histones in vitro, but, if X is normally lysosomal in distribution, then it probably is not a histone kinase. One of the great signalling spaghetti junctions is the Rho family of small GTPases, whose numerous members appear to regulate a bewildering plethora of cellular phenomena. Fortunately, some radical conceptchanging has been going on here too.

It has been known for some time that Rho proteins are recruited to the plasma membrane during activation. Now, however, one group has added an extraordinary dimension to this. Using a combination of immunoblotting and electron-microscopy techniques, Michaely et al.1 have shown that endogenous RhoA and Rac1 are highly abundant in caveolae, and that this enrichment is increased dramatically upon stimulation with platelet-derived growth factor (PDGF) or by ‘loading’ these GTPases with GTPgS (which forces them into an active state). Intriguingly, C3 toxin (which ribosylates and inactivates Rho) does not affect recruitment or targeting of GTPgS-loaded RhoA to caveolae but, rather, prevents it from being retained at this site. Since ribosylated

trends in CELL BIOLOGY (Vol. 9) November 1999

RhoA is inactive, it is tempting to suggest that retention in caveolae is therefore an essential element of Rho activation. Perhaps caveolae act to restrict diffusion along the membrane, pushing the quantity of ‘local’ signal above some critical threshold. The investigation of signalling from a ‘where’ rather than a ‘how’ perspective is a refreshing approach. Perhaps this is one subtle change that we should all adopt at an unsubtle pace.

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Michaely, P. A. et al. (1999) Polarized distribution of endogenous Rac1 and RhoA at the cell surface, J. Biol. Chem. 274, 21430–21436

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