Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Gene
2002 Jan 23;2831-2:299-309. doi: 10.1016/s0378-1119(01)00869-1.
Show Gene links
Show Anatomy links
Molecular cloning of XTP, a tau-like microtubule-associated protein from Xenopus laevis tadpoles.
Olesen OF
,
Kawabata-Fukui H
,
Yoshizato K
,
Noro N
.
???displayArticle.abstract???
The microtubules of the mammalian nervous system are stabilised by several microtubule-associated proteins (MAPs), including the tau and MAP-2 protein families. The most prominent feature of mammalian tau and MAP-2 proteins is a common and highly homologous microtubule-binding region consisting of three or four imperfect tandem repeats. In this paper we report the cloning and characterisation of a Xenopus laevis tau-like protein (XTP) from tadpole tails. This protein encompasses two isoforms of 673 or 644 amino acids with four tandem repeats that are highly homologous to mammalian tau repeats. Both isoforms share a common amino terminal half, whereas the carboxyl terminus downstream of the repeat region is unique for each isoform. Northern blot analysis revealed that both isoforms are preferentially expressed in the tail of X. laevis tadpoles, whereas a shorter version of XTP is expressed in the head. Recombinant proteins of both XTP isoforms were able to bind microtubules. The longest isoform, however, was more effective at promoting tubulin polymerisation, indicating that sequences downstream of the repeat region affect the microtubule assembling capacity. These results demonstrate that tau-like proteins are found in non-mammalian vertebrate species, where they may support the stability of microtubules.
Fig. 1. Sequence of XTP-1. Nucleotide and corresponding amino acid sequence of XTP-1 (GenBank accession number AY032847). The nucleotides are
numbered from the 50-end, and the first nucleotide in the initiation codon is shown in bold. The amino acid sequence is shown in single-letter code above the
nucleotide sequence, and the asterisk denotes the termination codon. The numbers on the right designate the position of nucleotides or amino acids. The amino
acid sequence unique to XTP-1 is shown in bold.
Fig. 2. Sequence of XTP-2. Nucleotide and corresponding amino acid sequence of XTP-2 (GenBank accession number AY032848). The nucleotides are
numbered from the 50-end, and the first nucleotide in the initiation codon is shown in bold. The amino acid sequence is shown in single-letter code above the
nucleotide sequence, and the asterisk denotes the termination codon. The numbers on the right designate the position of nucleotides or amino acids. The
truncated carboxyl terminal region, which differs from XTP-1, is shown in bold. The 124 bp insert in the nucleotide sequence, which causes the frameshift, is
underlined.
Fig. 3. Sequence homology between XTP and microtubule binding proteins from other species. Comparison of the repeat region from XTP with microtuble
associated proteins from other species. (A) Alignment of the four repeats of XTP with those of mammalian tau proteins (human, bovine, rat and mouse),
mammalian MAP-2 (human and rat), mammalian MAP-4 (human and mouse) and invertebrate PTL-1 (C. elegans). Residues that are conserved in at least six
of the ten different species of microtubule associated proteins are highlighted in bold. Dashes denote gaps in sequences that were introduced to obtain the
highest possible homology. (B) Homology of the repeat region between amino acid sequences of XTP and other types of microtubule associated proteins, as
represented by human tau, MAP-2, -4 and invertebrate PTL-1. C, Alignment of the proline rich regions that precede the tandem repeats in both XTP and human
tau. Conserved amino acid residues are underlined. Dashes denote gaps in sequences that were introduced to obtain the highest possible homology.
Fig. 4. Spatial expression of XTP mRNA. Northern blot analysis of RNA
from the head and the tail of Xenopus laevis tadpoles. A set of 2 mg of
poly(A)1 RNA from the head (H) and tail (T) of tadpoles at stage 54, 57,
and 61 was blotted. The blot was hybridised with probes specific for either
the amino terminal half (N-Probe) or the tandem repeat region of XTP (CProbe).
(A) Schematic representation of XTP with localisation of the Nprobe
(hatched box), C-probe (vertical lined box) and the carboxyl terminal
region unique to each isoform of XTP (black box); and (B) Northern blot
analysis of mRNA. The size of radioactive bands was calculated using a
standard RNA marker; (C) visual quantification of the relative expression
levels in tail and head of XTP mRNA as deducted from the Northern blot.
The expression level at stages 54–61 was averaged, with ‘ 1 ’ denoting a
very weak expression, whereas ‘1111’ denotes a high expression.
Fig. 5. Recombinant expression of XTP. (A) The purified recombinant proteins were separated by electrophoresis and visualised by staining with Coomassie
brilliant blue. Molecular weight standards are shown on the far left. Lane 1, human recombinant tau with 441 amino acids; lane 2, recombinant XTP-1, and lane
3, recombinant XTP-2. (B) Immunoblot of the recombinant proteins with a polyclonal antibody directed towards the carboxyl terminal half of human tau. Lane
1, XTP-1; lane 2, XTP-2, and lane 3, human tau with 441 amino acids. C: Immunoblot of the recombinant proteins with a polyclonal antibody, HM-2, directed
towards mammalian MAP-2. Lane 1, XTP-1; lane 2, XTP-2, and lane 3, human recombinant MAP-2d.
Fig. 6. Microtubule assembling capacity of XTP. Recombinant XTP proteins were incubated at 378C with tubulin monomers in micrototer plates, allowing ten
individual polymerisation reactions at six different concentrations to be measured simultaneously. Microtubule assembly was monitored by measuring changes
in turbidity at 405 nm. (A) The typical polymerisation of tubulin in the presence of 6 mM XTP-1 and -2 is shown. No change in turbidity was observed when
tubulin monomers were incubated in the absence of XTP protein. (B) The rate of polymerisation in the presence of various amounts of recombinant proteins
was monitored and averaged. The polymerisation is represented as the Vmax (i.e. maximum DOD/min) value (ordinates) against each tau concentration
(abcissa).
Fig. 7. Microtubule binding capacity of XTP. (A) To visualise the localisation of XTP in living cells, a cDNA coding for XTP-1 tagged with EGFP was
transiently transfected into COS cells using lipofectamine. After 48 h the cells were examined by fluorescence microscopy (magnification £ 750) to reveal the
distribution of XTP-EGFP. (B) Coomassie blue-stained gel of samples from microtubule binding experiment. Microtubules were incubated with 6 mM of
recombinant XTP for 15 min, and subsequently sedimented through a glycerol cushion by ultracentrifugation. Lane 1: XTP-1, pellet; lane 2: XTP-2, pellet; lane
3: XTP-1, supernatant, and lane 4: XTP-2, supernatant. XTP (upper band) was only present in lanes 1 and 2, indicating that it co-sedimented with microtubules
in the pellets.
