MOLECULAR ANALYSIS OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE IN TRYPANOPLASMA-BORELLI - AN EVOLUTIONARY SCENARIO OF SUBCELLULAR COMPARTMENTATION IN KINETOPLASTIDA

In Trypanoplasma borelli, a representative of the Bodonina within the Kinetoplastida, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activ ity was detected in bath the cytosol and glycosomes. This situation is similar to that previously found in Trypanosomatidae, belonging to a different Kinetoplastida suborder. In Trypanosomatidae different isoen zymes, only distantly related, are responsible for the activity in the two cell compartments. In contrast, immunoblot analysis indicated tha t the GAPDH activity in cytosol and glycosomes of T. borelli should be attributed to identical or at least very similar proteins related to the glycosomal GAPDH of Trypanosomatidae. Moreover, only genes related to the glycosomal GAPDH genes of Trypanosomatidae could be detected. All attempts to identify a gene related to the one coding for the tryp anosomatid cytosolic GAPDH remained unsuccessful. Two tandemly arrange d genes were found which are 95% identical. The two encoded polypeptid es differ in 17 residues. Their sequences are 72-77% identical to the glycosomal GAPDH of the other Kinetoplastida and share with them some characteristic features: an excess of positively charged residues, spe cific insertions, and a small carboxy-terminal extension containing th e sequence -AKL. This tripeptide conforms to the consensus signal for targeting of proteins to glycosomes. One of the two gene copies has un dergone some mutations at positions coding for highly conserved residu es of the active site and the NAD(+)-binding domain of GAPDH. Modeling of the protein's three-dimensional structure suggested that several o f the substitutions compensate each other, retaining the functional co enzyme-binding capacity, although this binding may be less tight. The presented analysis of GAPDH in T. borelli gives further support to the assertion that one isoenzyme, the cytosolic one, was acquired by hori zontal gene transfer during the evolution of the Kinetoplastida, in th e lineage leading to the suborder Trypanosomatina (Trypanosoma, Leishm ania), after the divergence from the Bodonina (Trypanoplasma). Further more, the data clearly suggest that the original GAPDH of the Kinetopl astida has been compartmentalized during evolution.