Identification and characterization of LFD1, a novel protein involved in membrane merger during cell fusion in Neurospora crassa

Abstract

SummaryDespite its essential role in development, molecular mechanisms of membrane merger during cell-cell fusion in most eukaryotic organisms remain elusive. In the filamentous fungus Neurospora crassa, cell fusion occurs during asexual spore germination, where genetically identical germlings show chemotropic interactions and cell-cell fusion. Fusion of germlings and hyphae is required for the formation of the interconnected mycelial network characteristic of filamentous fungi. Previously, a multipass membrane protein, PRM1, was characterized and acts at the step of bilayer fusion in N. crassa. Here we describe the identification and characterization of lfd-1, encoding a single pass transmembrane protein, which is also involved in membrane merger. lfd-1 was identified by a targeted analysis of a transcriptional profile of a transcription factor mutant (Δpp-1) defective in germling fusion. The Δlfd-1 mutant shows a similar, but less severe, membrane merger defect as a ΔPrm1 mutant. By genetic analyses, we show that LFD1 and PRM1 act independently, but share a redundant function. The cell fusion frequency of both Δlfd-1 and ΔPrm1 mutants was sensitive to extracellular calcium concentration and was associated with an increase in cell lysis, which was suppressed by a calcium-dependent mechanism involving a homologue to synaptotagmin.

Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

“…In addition to defects in membrane merger, the absence of PRM1 or LFD-1 also led to significant cell lysis of adhered germlings. The cell lysis defect in ⌬Prm1 and ⌬lfd-1 cells was suppressed by the addition of extracellular calcium and was shown to be dependent upon SYT1, a synaptotagmin homolog involved in calcium-mediated membrane damage repair (22). These observations suggest that membrane damage is associated with membrane merger and that PRM1 and LFD-1 play a role in preventing catastrophic membrane damage during cell fusion in N. crassa.…”

“…Strains constructed for this study are listed in Table 1. To obtain ⌬lfd-2 strains expressing cytoplasmic markers, the his-3 A strain (FGSC 6103) was used as the female in crosses with ⌬lfd-2 a. Progeny carrying the deletion mutation and the his-3 marker were used for transformation, with either pMF272 (30,31) or the modified version of pMF272 containing mCherry instead of green fluorescent protein (GFP), as described in Palma-Guerrero et al (22). Macroconidia were transformed by electroporation as described by Margolin et al (32).…”

“…The plasmid containing sec61-mCherry under the ccg-1 promoter was constructed by introducing the ORF of sec61 (NCU08897) into the modified version of pMF272 containing mCherry instead of GFP, as described in Palma-Guerrero et al (22), using the XbaI/PacI sites.…”

“…Strains were grown on Vogel's minimal media (VMM) (29) slant tubes for 4 to 6 days or until significant conidiation occurred. Conidial suspensions were prepared as described in Palma-Guerrero et al (22). Homokaryotic deletion strains were obtained via microconidiation selection (Table 1) (26,27).…”

“…PRM1 is the ortholog of S. cerevisiae Prm1p, and N. crassa ⌬Prm1 mutants have a defect in membrane fusion similar to that observed for S. cerevisiae prm1⌬ mutants (21). LFD-1 was recently identified as playing a role in membrane merger by screening for genes regulated by PP-1, a transcription factor that regulates genes associated with cell fusion in N. crassa (22,23). In addition to defects in membrane merger, the absence of PRM1 or LFD-1 also led to significant cell lysis of adhered germlings.…”