Sequence and structure of mtr, an amino acid transport gene, of Neurospora crassa

Abstract

The gene product of the mtr locus of Neurospora crassa participates in the transport of neutral aliphatic and aromatic amino acids across the plasma membrane. Mutants of the mtr locus have been identified by other workers by selecting for resistance to the poisonous amino acid analog 4-methyltryptophan. Using a transformation and selection scheme. three cosmids were cloned which complement the mtr-6(r) mutant allele. Two of the cloned DNAs were found to be tightly linked to restriction fragment length polymorphisms (RFLPs) which flank the mtr locus on linkage group IV. The mtr- gene was subcloned as a 2.9 kbp fragment; nested deletion and restriction site subclones of the fragment were generated and used to determine the DNA sequence. DNA sequence analyses indicated the presence of a 845 bp open reading frame (ORF) with a 59 bp intron. The proposed mtr ORF contains sequences which are 100% homologous to N. crassa consensus sequences at the ATG start site, at the exon/intron splice boundaries. and internal intron sequences. The mtr ORF is followed downstream by a possible polyadenylation signal. The 2.9 kbp fragment hybridizes to a poly A+ mRNA transcript of 2,300 nt. 51 nuclease mapping analysis of the transcript confirms the size of the transcript and the presence of the intron. An upstream open reading frame (uORF), found in the same reading frame as the mtr ORF, is proposed to be present in the mRNA transcript. The proposed mtr ORF is predicted to translate into a 261 amino acid polypeptide with a molecular weight of 28,613 daltons. Codon usage of the mtr polypeptide is biased with 18 of the 61 sense codons being absent. The polypeptide is composed of 164 non-polar amino acid residues which represent 63% of the amino acids. Hydropathy analysis' of the mtr polypeptide indicates six possible transmembrane spanning domains with an average length of 23 amino acids which are proposed to span the membrane in alpha helical conformations. The mtr polypeptide does not have a signal sequence and the polypeptide is proposed to function as a dimeric protein. Homology to a RNP octamer motif supports the proposal that the mtr polypeptide functions as a ribonucleoprotein (RNP) particle.