Characterization of the glyceraldehyde‐3‐phosphate dehydrogenase gene family from Kluyveromyces marxianus—polymerase chain reaction–single‐strand conformation polymorphism as a tool for the study of multigenic families

Abstract

Three glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Kluyveromyces marxianus were identified and characterized. The coding region of two of them (GAP2 and GAP3) is very similar (99.6% homology). The other gene (GAP1) is only 86% homologous to GAP2 or GAP3 and is responsible for the expression of Gap1p. This protein is extremely homologous to the K. marxianus cell wall protein p37, presumably involved in flocculation. However, no leader sequence could be detected in this gene. The identification of the three genes was possible with the use of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), as it permits us to overcome the difficulties caused by the high homology amongst the genes. Expression of the GAPDH genes under different carbon sources (glucose or ethanol) was assessed either by Northern blot or reverse transcription-PCR-SSCP analysis, revealing that genes GAP1 and GAP2, but not GAP3, are transcribed. The results also indicate that the transcription of the gene encoding the cell wall protein p37 (Gap1p) is not dependent on the carbon source, in contrast with the expression of the gene GAP2, which is affected in cells growing in a glucose-depleted medium.

The Crystal and Solution Structures of Glyceraldehyde-3-phosphate Dehydrogenase Reveal Different Quaternary Structures

“…Although kmGAPDH1p is detected at the cell surface of flocculent K. marxianus cells, it does not contain an N-terminal ER-targeting peptide (36). The evidence accumulated during the past decade showing that several proteins lacking an N-terminal signal peptide reach the yeast cell wall has led to the suggestion that an alternative ER-independent mechanism is responsible for the secretion of these proteins (60).…”

“…Subsequently the same observation was described for S. cerevisiae (38). Although kmGAPDH1p is detected at the cell surface and is N-glycosylated (37,39), it does not contain the N-terminal signal peptide (36). Sequence comparison shows that this protein is 81.6% identical to GAPDH isoform 1 from S. cerevisiae.…”

“…Of the three genes coding for GAPDH-like polypeptides, only GAP1 and GAP2 are transcribed (36). GAPDH isoform 1 from K. marxianus (kmGAPDH1p), formerly referred to as p37 (37), was first identified at the cell surface of flocculent K. marxianus cells, which was the first report of a non-cytosolic localization of a glycolytic enzyme.…”

Analysis of Expression of a Cytosolic Enzyme on the Surface of Streptococcus pyogenes

“…A number of potential explanations have been offered to account for the ability of these enzymes to associate with the cell membrane, including the secretion of the enzyme followed by the subsequent capture and binding by a cell surface structure (14) or a gene duplication event in which cytosolic proteins acquired appropriate signal and membrane anchoring sequences (1).…”

“…Increasingly, normally cytosolic enzymes associated with intracellular metabolism have been reported as surface constituents of both eukaryotic and prokaryotic cells (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Initially, these observations were considered potential artifacts due to the possible copurification of cytosolic materials during preparation of membrane fractions.…”