C-Terminal Signals Regulate Targeting of Glycosylphosphatidylinositol-Anchored Proteins to the Cell Wall or Plasma Membrane in Candida albicans

Abstract

Fungal glycosylphosphatidylinositol (GPI)-anchored proteins localize to the plasma membrane (PM), cell wall (CW), or both. To study signals that regulate PM versus CW targeting in Candida albicans, we (i) fused the N and/or C termini of the GPI CW protein Hwp1p and the GPI PM protein Ecm331p to green fluorescent protein (GFP) and (ii) expressed and localized the resulting fusions. Forty-seven amino acids from the C terminus of Hwp1p were sufficient to target GFP to the CW, and 66 amino acids from the C terminus of Ecm331p were sufficient to target GFP to the PM. Truncation and mutagenesis studies showed that G390 was the cleavage site in Ecm331p. Domain exchange and mutagenesis studies showed that (i) the 5 amino acids immediately N-terminal to the sites (the ؊ 5 to ؊ 1 amino acids) played key roles in targeting to the PM or CW; (ii) KK and FE residues at positions ؊ 1 and ؊ 2, respectively, targeted to the PM and CW; and (iii) a loss of I at position ؊ 5 increased PM retention. Small fluorescent reporters can be used to study the peptide signals that regulate PM versus CW targeting of GPI proteins and may be useful for identifying proteins that interact with key targeting signals.

The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity

“…1B, C). The amino acids upstream of the v sites were also conserved, and the absence of a dibasic motif and occurrence of an acidic residue suggested a cell wall location for both Pga59 and Pga62 (Caro et al, 1997;Mao et al, 2008). Pga62 differed from Pga59 by the occurrence of an additional 42 aa repeat (Fig.…”

“…In the case of C. albicans, localization of GPI-modified proteins appears to be dependent on similar as well as additional determinants (Richard & Plaine, 2007). For instance, Mao et al (2008) have shown that the occurrence of an aromatic residue and an acidic residue in positions v-2 and v-1, respectively, promotes cell wall localization of C. albicans GPI-modified proteins. There are 115 putative GPI-modified proteins in C. albicans and most of them have unknown functions.…”

Unexpected Role for a Serine/Threonine-Rich Domain in the Candida albicans Iff Protein Family

“…We have name these proteins GPI-anchored cell wall proteins hereafter in the text. Several studies have attempted to identify the signal governing the final localization of GPI-anchored proteins, and they pointed out the crucial role of basic amino acids upstream of the GPI anchor attachment site (the site) for plasma membrane retention (8,24,25,29,47).…”

Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

“…These experiments identified the omega cleavage site. Further domain exchange and mutagenesis studies identified which residues dictated cell wall or plasma membrane localization (Mao et al, 2008).…”

“…Site-directed mutagenesis has been used to determine how GPI-tagged proteins discriminate between localization to the plasma membrane and cell wall (Mao et al, 2008). N and C termini of cell wall or plasma proteins were fused to GFP.…”