APT1, but not APT2, codes for a functional adenine phosphoribosyltransferase in Saccharomyces cerevisiae

Abstract

The yeast Saccharomyces cerevisiae has two separate genes (APT1 and APT2) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT). However, genetic analysis indicated that only APT1 could code for a complementing activity. Cloning and expression of both the APT1 and APT2 genes in Escherichia coli showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity. Northern and Western blot analyses demonstrated that only APT1 was transcribed and translated under normal physiological conditions in yeast. Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event. We conclude that APT1 is the functional gene in S. cerevisiae and that APT2 is a pseudogene.

FIG. 1

Expression of hexahistidine-tagged APRT1 and APRT2 proteins in E. coli cells. Lane 1, total cell extract from E. coli cells transformed with the His-tagged APT1 gene; lane 2, Ni²⁺-purified recombinant APRT1 protein; lane 3, total cell extract from E. coli cells transformed with the His-tagged APT2 gene; lane 4, Ni²⁺-purified recombinant APRT2 protein; lanes 5 and 6, purified recombinant APRT1 and APRT2 proteins (respectively) run next to each other to emphasize their relative size difference.

FIG. 2

Sizing of the APRT1 and APRT2 recombinant proteins. (A) His-tagged APRT1 was chromatographed on a Sephadex G-200 column as described in Materials and Methods. The peak of activity corresponds to that of a 50-kDa protein. (B) The His-tagged APRT2 protein was chromatographed through the same Sephadex column. As no APRT activity was detected with this protein, the peak of absorbance (optical density at 280 nm [O.D.280]) is given and corresponds to that of a 26-kDa protein. β-Amylase (200 kDa), bovine serum albumin (BSA; 66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (cyt C; 13 kDa) were used as standards to calibrate the column. vo, void volume; vt, total volume.

FIG. 3

Western blot analysis of native APRT proteins. (A) Western blot probed with polyclonal anti-APRT1 antibody. Lane 1 contains a cell extract from strain Y350 (wild-type strain), lane 2 contains a cell extract from strain Y511 (apt1 mutant strain), and lane 3 contains pure recombinant APRT1. (B) Western blot similar to that shown in panel A but probed with polyclonal anti-APRT2 antibody. Lanes 1 and 2 contain cell extracts from strains Y350 and Y511 (respectively), and lane 3 contains pure recombinant APRT2.

FIG. 4

Northern blot analysis of the APT1 and APT2 gene products from strain X79 (wild type). (A) Total RNA was extracted at various times during growth and probed with a radiolabeled APT1 probe. (B) Same membrane as that shown in panel A, probed with a radiolabeled APT2 probe. (C) Same membrane as that shown in panel A, probed with a radiolabeled yeast actin used as a loading control.

FIG. 5

Comparative phylogenetic reconstruction of APRT1 and APRT2. A phylogenetic tree of various deduced APRT sequences constructed with PAUP version 3.11 is shown. Numbers indicate the probability of two sequences branching together.