latrunculin-a and lucifer-yellow

The function of endocytic pathway in filamentous fungi has remained elusive. Recently, we have identified that FgEnd1, which has a 27% amino acid homology and shares specific EH3 domain with ScEnd3 of Saccharomyces cerevisiae, is a putative member of the endocytic machinery in Fusarium graminearum. The failure of the scend3 mutant to uptake Lucifer yellow (LY) was recovered by introducing FgEnd1 into S. cerevisiae. The deletion of fgend1 in F. graminearum resulted in a 2-fold decrease in the rate of uptake of the endocytic marker FM4-64 when compared to wild-type cells. The rate of uptake was similar to that seen in latrunculin A (Lat-A)-treated cells. Furthermore, fgend1 deletion strain of F. graminearum showed lower ferrichrome (FC) uptake activity than wild-type F. graminearum, and the same rate as LatA-treated cells. Taken together, these results suggest that FgEnd1 is a putative member of the endocytic machinery, although it acts through a different mechanism from ScEnd3 or ScEnd4 of S. cerevisiae.

Topics: Amino Acid Sequence; Bridged Bicyclo Compounds, Heterocyclic; Coloring Agents; Cytoskeletal Proteins; Endocytosis; Ferrichrome; Fungal Proteins; Fusarium; Isoquinolines; Molecular Sequence Data; Protein Structure, Tertiary; Pyridinium Compounds; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Thiazolidines

Herein we report that Gts1p fused with green-fluorescent protein (GFP) is localized in the cortical actin patch besides nuclei in yeast and the cortical Gts1p changed its position together with the patch depending on the cell-cycle phase, while nuclear Gts1p accumulated predominantly in the budding phase. Whereas Gts1p does not directly bind to actin, it associated mainly with the actin-associated protein Pan1p. In the GTS1-deleted transformant gts1Delta, the number of cells containing either a fragmented vacuole or an enlarged single central vacuole increased and the uptake of the hydrophilic dye Lucifer yellow (LY) in the vacuole decreased. Further, gts1Delta transformed with a mutant Gts1p having two cysteine-to-alanine substitutions in a zinc finger resembling that of GTPase-activating proteins of ADP-ribosylation factors (ARF-GAP) neither recovered the LY uptake unlike gts1Delta transformed with the wild-type GTS1, nor reduced the average size of central vacuoles as much as the latter did. These results suggested that Gts1p in the actin patch is involved in the fluid-phase endocytosis and membrane trafficking for vacuole formation and that the putative ARF-GAP domain in Gts1p plays an important role in these functions.

Topics: Actins; ADP-Ribosylation Factors; Amino Acid Sequence; Bridged Bicyclo Compounds, Heterocyclic; Endocytosis; Gene Deletion; Green Fluorescent Proteins; GTPase-Activating Proteins; Immunoprecipitation; Isoquinolines; Molecular Sequence Data; Protein Structure, Tertiary; Protein Transport; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Subcellular Fractions; Thiazolidines; Transcription Factors; Vacuoles