Structure and regulated expression of genes encoding fructose biphosphate aldolase in Trypanosoma brucei.

Abstract

Low stringency hybridisation with a rabbit aldolase cDNA was used to select cDNA clones encoding fructose biphosphate aldolase in Trypanosoma brucei. A clone which is almost full length encodes a protein of 41 027 daltons which has 50% identity with rabbit aldolase A and slightly lower homology with B‐type aldolases. The homologous mRNA is at least 6‐fold more abundant in bloodstream trypomastigotes than in procyclic forms, as expected from measurements of enzyme activity. Genomic mapping results indicate that trypanosomes have four copies of the aldolase gene arranged as two copies of a tandem repeat. The protein has a short N‐terminal extension (relative to other known aldolases) which could be involved in the glycosomal localisation of the enzyme.

Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties

“…The cDNA for glycosomal aldolase of T. brucei predicts a subunit mass of 40.967 kDa [29]. This value is in perfect agreement with our value for the purified enzyme as obtained from SDS gels (40.5 f 0.5 kDa).…”

“…In this respect it is highly relevant that the glycosomal enzymes are basic proteins and have PI values which are 1 -6 higher than those found for other organisms. This results in highly positively charged proteins (for instance, a net charge of + 13 for glycosomal phosphoglycerate kinase [27] and + 10 for aldolase [29]) and such a positive charge or charges could play an important role in the recognition of the enzyme by, or its entry into, the organelle. Although we cannot exclude that the above-described unique properties of the glycosomal enzymes play a role in their proper functioning inside the glycosome, we now strongly believe that we are dealing here with signals for import.…”

In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.

“…However, of the nine glycosomal enzymes sequences that are known, T. brucei fructose biphosphate aldolase (Clayton, 1985), T. brucei triosephosphate isomerase (Swinkels et al, 1986), T. brucei phosphoenolpyruvate carboxykinase (Kueng et al, 1989;Parsons and Smith, 1989), as well as a minor glycosomal phosphoglycerate kinase encoded by the A gene of the T. brucei PGK gene cluster (Alexander et al, 1990), still do not contain an obvious C-terminal tripeptide sequence that might function in glycosomal import by our present findings. These proteins may rely on a somewhat different import mechanism.…”

Function of N‐terminal import signals in trypanosome microbodies

“…Indeed, the presence of a C-terminal signal may be precluded by strict catalytic requirements at the extreme C-terminus [23]. Comparison of trypanosome aldolase with mammalian aldolases (which are of course cytoplasmic) revealed the presence of a trypanosome-specific N-terminal extension [18,24]. An alignment of this sequence with known or predicted PTS-2 sequences is shown in Fig.…”

“…To construct the expression vector pAldE a PCR was performed with pAldl7 [18] as template, The primers CZ031 5'TTCAC-AAGCTTCACAATGTCCAAGCGTGTTGAAGTT3' and CZ049 5'ATATCCAGTGATTTTTTTCTCGAGCGCTTCATATGGCGTC-TTCAG3' (restriction sites underlined) were used to amplify a fragment encoding the 24 N-terminal amino acids of aldolase joined to the first six amino acids of CAT starting with glutamic acid (the amino acid immediately following the initiator methionine), pJP44 [19] was used as template in a second PCR to amplify the CAT gene with the primers CZ030 5'CTGAAGACGCCATATGAAGCGCTCGAG-AAAAAAATCACTGGATAT3' and CC12 5'GTTTCGTTCCTCC-GAGGCGC3'. The product is a CAT coding region that can hybridise to the first via a junction segment containing an XhoI site.…”