Figure 2.
epg-6 Encodes a WD40 Repeat PtdIns(3)P-Binding Protein that Is Widely Expressed (A)
epg-6 maps to linkage group III (LG III), around +3.2. (B) Protein sequence of EPG-6. Mutations identified in
epg-6 mutants are shown in red. The seven putative b-propeller blades are underlined. In the EPG-6a isoform, amino acids 331-333 (IPE, highlighted in red) are replaced by K. (C) Alignment of the fifth and sixth b-propeller blades of ATG-18, EPG-6, yeast Atg18 and Atg21, and human WIPI1 and WIPI4. The FRRG and LRRG motifs are in red. (D and E) Accumulation of GFP::PGL-1 granules in somatic cells in
epg-6 mutant embryos. Nomarski image (D) of the embryo shown in (E). (F) A hs::WIPI4 transgene suppresses accumulation of GFP::PGL-1 in somatic cells in
epg-6 mutants. (G) gfp::
epg-6 is widely expressed during embryogenesis. An 200 cell embryo is shown. (H and I) GFP::PGL-1 granules accumulate in somatic cells in
atg-18 mutant embryos. (H): Nomarski image of the embryo shown in (I). (J and K) The hs::
atg-18 transgene (J), but not the hs::
atg-18(FTTG) transgene (K), rescues defective degradation of PGL granules in
atg-18 mutants. (L) A large number of SEPA-1::GFP aggregates are formed in late-stage
epg-6 mutant embryos. (M) The transgene containing hs::
epg-6(del 6), in which the sixth b-propeller blade of EPG-6 is deleted, fails to rescue defective degradation of SEPA-1::GFP in
epg-6 mutants. (N) SEPA-1 aggregates are not detected in wild-type embryos at the 2-fold stage. (O) SEPA-1 aggregates accumulate in 2-fold stage embryos overexpressing the fifth and sixth blades of EPG-6. (P) Binding of wild-type and mutated EPG-6 and ATG-18 to various lipids. EPG-6(del 5+6), EPG-6 with a deletion of the fifth and sixth b-propeller blades; EPG- 6(5+6), fragment of EPG-6 containing only the fifth and sixth b-propeller blades; LPA, lysophosphatidic acid; LPC, lysophosphocholine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.