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. 2002 Jan;184(2):427-32.
doi: 10.1128/JB.184.2.427-432.2002.

Respiration-dependent utilization of sugars in yeasts: a determinant role for sugar transporters

Paola Goffrini  1 , Iliana Ferrero, Claudia Donnini
Affiliations

Respiration-dependent utilization of sugars in yeasts: a determinant role for sugar transporters

Paola Goffrini et al. J Bacteriol. 2002 Jan.

Abstract

In many yeast species, including Kluyveromyces lactis, growth on certain sugars (such as galactose, raffinose, and maltose) occurs only under respiratory conditions. If respiration is blocked by inhibitors, mutation, or anaerobiosis, growth does not take place. This apparent dependence on respiration for the utilization of certain sugars has often been suspected to be associated with the mechanism of the sugar uptake step. We hypothesized that in many yeast species, the permease activities for these sugars are not sufficient to ensure the high substrate flow that is necessary for fermentative growth. By introducing additional sugar permease genes, we have obtained K. lactis strains that were capable of growing on galactose and raffinose in the absence of respiration. High dosages of both the permease and maltase genes were indeed necessary for K. lactis cells to grow on maltose in the absence of respiration. These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in K. lactis and probably in many other yeasts.

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Figures

FIG. 1.
FIG. 1.
Growth curves of wild-type and cyt1 mutant strains in galactose. The wild-type strain JBD100 (wt) and its isogenic cyt1 mutant were transformed with either a multicopy GAL2 plasmid (pUK-GAL2) or a monocopy GAL2 plasmid (KCp-GAL2). The transformants were grown in glucose minimal medium to mid-exponential phase and then transferred to galactose complete medium (YPGal) with or without addition of 5 mM antimycin A (AA). The cultures were grown on a reciprocating shaker at 110 rpm. Growth was followed for 66 h by A600 measurements. Growth curves of nontransformed strains are included for comparison.
FIG. 2.
FIG. 2.
Effect of antimycin A on galactose transport. K. lactis cells (PM4-4B), transformed or not with GAL2-carrying monocopy plasmid KCp-GAL2 and with GAL2-carrying multicopy plasmid pUK-GAL2, were grown on galactose, washed, and suspended in phosphate buffer. Uptake radiolabeled galactose was determined in the presence and absence of antimycin A as detailed in Materials and Methods. Time zero values were subtracted from all measurements. Symbols: wild-type PM4-4B in the absence (bull;) and presence (○) of antimycin A; PM4-4B/KCp-GAL2 in the absence (▪) and presence (□) of antimycin A; PM4-4B/pUK-GAL2 in the absence (▴) and presence (▵) of antimycin A. The values are means of three independent experiments. In no case was the variation higher than 15%.
FIG. 3.
FIG. 3.
(A) Physical map of the HXT4 gene region of S. cerevisiae chromosome VIII. The region cloned into the K. lactis transformant clone F (see text) is shown. The flanking sequences used as primers for sequencing are indicated with arrows and broken lines. pAF1 and pKS1 are subclones. (B and C) Physical map and subcloning of the MAL regions of S. cerevisiae and K. lactis, respectively. Plasmid pCV-H2 contains S. cerevisiae Mal6T (maltose permease) and MAL6S (maltase), and plasmid pEFPH contains the K. lactis MAL22 gene (maltase) (A. Dominguez, unpublished data). pKK1, pKL1, pMM1, pKM1, and pUK-MAL22 are subclones. Open reading frames and DDSE sequences are shown by boxes. DNA inserts are represented by thin lines. Restriction sites: B, BglII; H, HindIII; P, PstI; X, XbaI. The + and − signs stand for the presence and absence, respectively, of transformed clones on raffinose plus antimycin A medium (A) and on maltose plus antimycin A medium (B and C).
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