Conservation of the WD-repeat, microtubule-binding protein, EMAP, in sea urchins, humans, and the nematode C. elegans.

The echinoderm microtubule-associated protein (EMAP) is the most abundant microtubule-binding protein in the first cleavage mitotic apparatus in sea urchin embryos. The first goal of this study was to determine whether there is sufficient EMAP in the egg and embryo to modify microtubule dynamics during the early cleavages divisions and whether EMAP functions at a specific time or place in the embryo. To accomplish this goal, we examined the relative abundance, tissue distribution, and temporal pattern of EMAP expression during embryonic development. The second goal of this study was to identify important functional domains within the EMAP coding sequence. A conserved sequence might reveal a potential microtubule-binding domain. We cloned, sequenced and compared overlapping EMAP cDNAs from two different sea urchin species that diverged approximately 80 million years ago, and compared these with cDNA sequences from a vertebrate and nematode species. From quantitative immunoblots, we determined the EMAP concentration in eggs to be 4 microM. The steady-state levels of EMAP mRNA and protein accumulated during development, and all three germ layers expressed EMAP. During the early stages of development, EMAP and tubulin were both abundant in the ectoderm, mesoderm and endoderm. However, during late gastrulation and the formation of the early pluteus larvae, EMAP was enriched in the mesoderm, while tubulin staining was most abundant in the archenteron. These results indicate that EMAP may have tissue-specific functions in the late stage embryo. To identify conserved functional domains, we compared the predicted amino acid sequence encoded by Strongylocentrotus purpuratus and Lytechinus variegatus EMAP cDNAs, and determined that these two sea urchin EMAPs were 95% conserved and shared an identical domain organization. A parsimonious analysis of these sea urchin protein sequences, as well as human and C. elegans EMAP sequences was used to construct a gene tree. Together these results suggest that EMAP is an important microtubule protein required at all developmental stages of sea urchins, and whose cellular function may be conserved amongst metazoans.

Sequence and expression patterns of a human EMAP-related protein-2 (HuEMAP-2).

Until recently, the microtubule-associated protein, EMAP, was identified only in echinoderms such as sea urchin, starfish and sand dollar. Sea urchin EMAP localizes to the mitotic apparatus in vivo and modifies the assembly dynamics of microtubules in vitro. To identify domains important for EMAP function, we cloned and sequenced cDNAs for an EMAP-related protein in human. The nucleotide sequence of a human EMAP-related protein-2 (HuEMAP-2) encodes a protein of 649 amino acids in length. The translated polypeptide sequence and domain structure of sea urchin EMAP and HuEMAP-2 are highly conserved, with greater than 57% identity and 77% similarity at the translated amino acid level. Southern blot analysis is consistent with the presence of a single HuEMAP-2 gene in the human genome. Moreover, HuEMAP-2 is a member of a larger protein family with at least four HuEMAP sequences in the NCBI databases. One of these, HuEMAP-1, is identified as the candidate gene for the Usher syndrome 1 a locus (Genomics 43:104-106, 1997). Northern blot analysis indicates that HuEMAP-1, and HuEMAP-2 are expressed in different human tissues. In addition, these RNA blots indicate that HuEMAP-2 transcripts may be differentially spliced in neuronal tissues.