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There is no definitive evidence on the nature of the cap at microtubule ends that is responsible for dynamic instability behavior. It was, therefore, of interest that steady-state microtubules assembled in 20 mM P(i) buffer and pulsed for 15-60 min with [gamma-(32)P]GTP contained approximately 26 [(32)P]P(i)/microtubule [Panda et al. (2002) Biochemistry 41, 1609-1617]. It was concluded that microtubules are capped with a tubulin-GDP-P(i) subunit at the end of each its 13 protofilaments and that this is responsible for stabilizing microtubules in the growth phase. Also, because microtubules with [(32)P]P(i) were isolated despite the presence of 20 mM P(i), it was concluded that P(i) in terminal tubulin-GDP-P(i) subunits does not exchange with solvent. These observations are inconsistent with our finding that tubulin-GDP-P(i) subunits do not stabilize microtubules and with evidence that the nucleotide, and presumably also P(i), in subunits at microtubule ends exchanges with solvent. We have resolved this discrepancy by finding that during the pulse period the added [(32)P]GTP was almost quantitatively hydrolyzed. The so-formed [(32)P]P(i) labeled the 20 mM P(i) buffer, and this exchanged into tubulin-GDP subunits in the core of the microtubule. Evidence for this was our finding of virtually identical [(32)P]P(i) in microtubules pulsed with [(32)P]GTP with a specific activity that varied 11-fold by using either 100 or 1,100 microM GTP in the reaction. Label uptake was insensitive to the [(32)P]GTP specific activity because in both cases hydrolysis generated 20 mM [(32)P]P(i) with a virtually identical specific activity. Also, approximately 0.4 mol of [(32)P]P(i) /tubulin dimer was found in microtubules when steady-state microtubules in 20 mM P(i) were pulsed with a trace amount of [(32)P]P(i). This stoichiometry is consistent with a 25 mM K(d) previously reported for P(i) binding to tubulin-GDP subunits in microtubules. It is concluded that, under the conditions used for the [(32)P]GTP pulse labeling, (32)P was incorporated into the entire microtubule from [(32)P]P(i) released into the solution, rather than into a tubulin-GDP-P(i) cap, from [(32)P]GTP. Thus, there is no evidence that tubulin-GDP-P(i) subunits accumulate in and stabilize microtubule ends.

Localization of maternal nanos mRNA to the posterior pole is essential for development of both the abdominal segments and primordial germ cells in the Drosophila embryo. Unlike maternal mRNAs such as bicoid and oskar that are localized by directed transport along microtubules, nanos is thought to he trapped as it swirls past the posterior pole during cytoplasmic streaming. Anchoring of nanosdepends on integrity of the actin cytoskeleton and the pole plasm; other factors involved specifically in its localization have not been described to date. Here we use genetic approaches to show that the Hsp90 chaperone (encoded by Hsp83 in Drosophila) is a localization factor for two mRNAs, nanos and pgc. Other components of the pole plasm are localized normally when Hsp90 function is partially compromised, suggesting a specific role for the chaperone in localization of nanos and pgc mRNAs. Although the mechanism by which Hsp90 acts is unclear, we find that levels of the LKB1 kinase are reduced in Hsp83 mutant egg chambers and that localization of pgc (bitt not nos) is rescued upon overexpression of LKB1 in such mutants. These observations suggest that LKB1 is a primary Hsp90 target for pgc localization and that other Hsp90 partners mediate localization of nos. [ABSTRACT FROM AUTHOR]