Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein.

Abstract

Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts. It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus. The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment. Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing. Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase. The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence. In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec6l and sec62 translocation mutants.Many specific yeast permeases have been subjects of genetic and physiological studies (8), while more recent reports have concentrated on their molecular biology (1,5,6,13,21,30,43,51). Protein structure prediction programs have been used to propose preliminary structural models based on the corresponding DNA sequences (21, 42, 51). The sequencing data indicate that several yeast sugar transporters are homologous with bacterial and mammalian sugar transporters (5, 6, 42), such as several human glucose transporters (27). The members of this sugar transporter superfamily all have the same general structure, consisting of 12 potential membrane-spanning segments connected by hydrophilic loops of similar relative lengths (42). The structures of these transporters differ mainly in the lengths of their N termini and to a lesser extent in the lengths of their C-terminal segments. The human glucose transporters have very short N termini (12 amino acids long) (27), as do the bacterial transporters (24), while the yeast members of this family have N termini of 66 (42) to 97 (5) amino acids. All of the other yeast permeases sequenced so far (1,21,30,43,51) have similarly long N-terminal hydrophilic extensions. The orientation of these segments with respect to the plasma membrane has not been demonstrated experimentally for these yeast proteins. A number of yeast permeases carry potential N glycosylation sites, but there is as yet no clear indication as to whether any of these permeases is indeed a glycoprotein. These proteins have not yet been analyzed biochemically and no yeast permease has been purified, whereas several mammalian glucose transporters have been purified (19). Purification and extensive biochemical studies have been performed on the Escherichia coli lactose permease (11, 48), for which overexpression of active protein could be obtained (44). In contrast, several attempts at overexpression of active yeast transporters ...

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