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Lipid phosphate phosphatases dimerise, but this interaction is not required for in vivo activity.

Burnett C, Makridou P, Hewlett L, Howard K - BMC Biochem. (2004)

Bottom Line: Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2.Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation.The results indicate that the associations we see are highly specific and occur only between monomers of the same protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, MRC Laboratory for Molecular Cell Biology, University College London, Gower St, London WC1E 6BT, UK. c.burnett@ucl.ac.uk

ABSTRACT

Background: Lipid phosphate phosphatases (LPPs) are integral membrane proteins believed to dephosphorylate bioactive lipid messengers, so modifying or attenuating their activities. Wunen, a Drosophila LPP homologue, has been shown to play a pivotal role in primordial germ cell (PGC) migration and survival during embryogenesis. It has been hypothesised that LPPs may form oligomeric complexes, and may even function as hexamers. We were interested in exploring this possibility, to confirm whether LPPs can oligomerise, and if they do, whether oligomerisation is required for either in vitro or in vivo activity.

Results: We present evidence that Wunen dimerises, that these associations require the last thirty-five C-terminal amino-acids and depend upon the presence of an intact catalytic site. Expression of a truncated, monomeric form of Wunen in Drosophila embryos results in perturbation of germ cell migration and germ cell loss, as observed for full-length Wunen. We also observed that murine LPP-1 and human LPP-3 can also form associations, but do not form interactions with Wunen or each other. Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2. Finally we discovered that addition of a trimeric myc tag to the C-terminus of Wunen does not prevent dimerisation or in vitro activity, but does prevent activity in vivo.

Conclusion: LPPs do form complexes, but these do not seem to be specifically required for activity either in vitro or in vivo. Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation. The results indicate that the associations we see are highly specific and occur only between monomers of the same protein.

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Proteins at the Cell Surface (a) Biotinylation of WunM3 and WunGFP at the cell surface. Washes = non-biotinylated proteins; Eluates = biotinylated cell surface proteins. We used internal tubulin as a negative control and the cell surface marker CD2 as a positive control (data not shown). (b) EM shows both proteins at the cell surface, with WunM3 present in sub-cortical vesicles.
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Figure 4: Proteins at the Cell Surface (a) Biotinylation of WunM3 and WunGFP at the cell surface. Washes = non-biotinylated proteins; Eluates = biotinylated cell surface proteins. We used internal tubulin as a negative control and the cell surface marker CD2 as a positive control (data not shown). (b) EM shows both proteins at the cell surface, with WunM3 present in sub-cortical vesicles.

Mentions: As it has been indicated that the LPPs may function as ecto-enzymes, we wanted to confirm that neither tag interfered with trafficking to the cell surface. We could then rule out the possibility that the M3 tag prevented WunM3 from reaching the cell surface precluding access to its substrate. We achieved this by incubating intact cells expressing either WunM3 or WunGFP with NHS-biotin – a cell impermeable biotin that labels only those proteins expressed on the cell surface – and running lysates over a over a monomeric biotin binding avidin column before Western blotting (Fig. 4a). Densitometry analysis by Bio-Rad Quantity One Software indicated that similar percentages of each protein were present at the cell surface (data not shown). Examination by electron microscopy (EM) shows WunGFP both on the cell surface and in large, internal, multi-vesicular structures, whereas WunM3 is found in both these locations and is also associated with vesicles just below the plasma membrane (Fig. 4b).


Lipid phosphate phosphatases dimerise, but this interaction is not required for in vivo activity.

Burnett C, Makridou P, Hewlett L, Howard K - BMC Biochem. (2004)

Proteins at the Cell Surface (a) Biotinylation of WunM3 and WunGFP at the cell surface. Washes = non-biotinylated proteins; Eluates = biotinylated cell surface proteins. We used internal tubulin as a negative control and the cell surface marker CD2 as a positive control (data not shown). (b) EM shows both proteins at the cell surface, with WunM3 present in sub-cortical vesicles.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC319698&req=5

Figure 4: Proteins at the Cell Surface (a) Biotinylation of WunM3 and WunGFP at the cell surface. Washes = non-biotinylated proteins; Eluates = biotinylated cell surface proteins. We used internal tubulin as a negative control and the cell surface marker CD2 as a positive control (data not shown). (b) EM shows both proteins at the cell surface, with WunM3 present in sub-cortical vesicles.
Mentions: As it has been indicated that the LPPs may function as ecto-enzymes, we wanted to confirm that neither tag interfered with trafficking to the cell surface. We could then rule out the possibility that the M3 tag prevented WunM3 from reaching the cell surface precluding access to its substrate. We achieved this by incubating intact cells expressing either WunM3 or WunGFP with NHS-biotin – a cell impermeable biotin that labels only those proteins expressed on the cell surface – and running lysates over a over a monomeric biotin binding avidin column before Western blotting (Fig. 4a). Densitometry analysis by Bio-Rad Quantity One Software indicated that similar percentages of each protein were present at the cell surface (data not shown). Examination by electron microscopy (EM) shows WunGFP both on the cell surface and in large, internal, multi-vesicular structures, whereas WunM3 is found in both these locations and is also associated with vesicles just below the plasma membrane (Fig. 4b).

Bottom Line: Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2.Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation.The results indicate that the associations we see are highly specific and occur only between monomers of the same protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, MRC Laboratory for Molecular Cell Biology, University College London, Gower St, London WC1E 6BT, UK. c.burnett@ucl.ac.uk

ABSTRACT

Background: Lipid phosphate phosphatases (LPPs) are integral membrane proteins believed to dephosphorylate bioactive lipid messengers, so modifying or attenuating their activities. Wunen, a Drosophila LPP homologue, has been shown to play a pivotal role in primordial germ cell (PGC) migration and survival during embryogenesis. It has been hypothesised that LPPs may form oligomeric complexes, and may even function as hexamers. We were interested in exploring this possibility, to confirm whether LPPs can oligomerise, and if they do, whether oligomerisation is required for either in vitro or in vivo activity.

Results: We present evidence that Wunen dimerises, that these associations require the last thirty-five C-terminal amino-acids and depend upon the presence of an intact catalytic site. Expression of a truncated, monomeric form of Wunen in Drosophila embryos results in perturbation of germ cell migration and germ cell loss, as observed for full-length Wunen. We also observed that murine LPP-1 and human LPP-3 can also form associations, but do not form interactions with Wunen or each other. Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2. Finally we discovered that addition of a trimeric myc tag to the C-terminus of Wunen does not prevent dimerisation or in vitro activity, but does prevent activity in vivo.

Conclusion: LPPs do form complexes, but these do not seem to be specifically required for activity either in vitro or in vivo. Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation. The results indicate that the associations we see are highly specific and occur only between monomers of the same protein.

Show MeSH
Related in: MedlinePlus