There was an error in Development (2018) 145, dev151571 (doi:10.1242/dev.151571).

The amino acid sequence reported as corresponding to the temperature-sensitive mutation in Drosophila Sac1, and tested for protein stability via tissue culture, was incorrect.

Rather than the amino acid substitution G382R, the point mutation in Sac1ts is predicted to lead to the amino acid substitution G382D. Because the cell culture protein stability assay conducted was carried using the incorrect sequence, experiments have been repeated to determine whether the actual amino acid substitution present in Sac1ts mutants (G382D) also destabilizes the Sac1 protein.

To do this, wild-type Sac1 was expressed, as well as mutant Sac1 proteins containing the actual temperature-sensitive (ts) substitution (G382D; now designated TS1) and the erroneous temperature-sensitive substitution (G382R; TS2). In this follow-up experiment, the various Sac1 proteins were expressed in Drosophila S2 cells and permissive and non-permissive temperatures suitable for these cells (18°C and 29°C, respectively) were used.

Site-directed mutagenesis was carried out using QuikChange II XL (Stratagene) to generate TS1 (G382D) and TS2 (G382R) alleles of Drosophila Sac1 (dSac1) from wild-type dSac1 cDNA in a pUC57 vector (BioBasics). dSac1 TS1 was generated using forward primer 5′-GTGTCCACGCAGACTGATGTCTTCCGAACG-3′ and reverse primer 5′-CGTTCGGAAGACATCAGTCTGCGTGGACAC-3′. dSac1 TS2 was generated using forward primer 5′-GTGTCCACGCAGACTCGTGTCTTCCGAACG-3′ and reverse primer 5′-CGTTCGGAAGACACGAGTCTGCGTGGACAC-3′.

Gateway technology was used to generate dSac1 constructs for expression in Drosophila S2 cells. dSac1 wild-type, TS1 and TS2 alleles were amplified using primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATGGACAGCAGGGAGGAGAAC-3′, reverse primer 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTGTCATGGGTCTCGAAAGGAGATGG-3′. PCR fragments were cloned into pDONR221 (ThermoFisher, 12536017) using a BP Clonase II reaction (ThermoFisher, 11789020) and then transferred into Drosophila Gateway vector pAHW (DGRC Stock 1095) using the Clonase LR II reaction (ThermoFisher, 11791020) to generate N-terminally 3xHA-tagged Act5C-driven protein expression plasmids.

S2 cells were grown in Schneider's Drosophila medium (ThermoFisher, 21720-024) supplemented with 10% fetal bovine serum. Cells were seeded in 24-well plates for 4 h before transfection and transfected with plasmids for transient expression of 3xHA-tagged dSac1 constructs using Effectene (Qiagen, 301427) as per manufacturer's instructions. Transfected cells were kept for 3 days at 18°C and then subsequently incubated for 10 min at either 18°C or 29°C. Cells were lysed in SDS loading buffer [final concentration 50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA and 0.02% Bromophenol Blue]. HA-tagged dSac1 proteins were analyzed by SDS PAGE and immunoblotting using rat anti-HA antiserum (1:5000; Sigma Aldrich, 11867423001). Anti-GAPDH antibody (1:5000; Sigma Aldrich, G9545) was used to quantify the loading control.

Densitometry values of the HA-Sac1 immunoreactive bands were determined using FIJI (ImageJ) version 1.0, and normalized to GAPDH.

As predicted, both mutations result in unstable proteins. Indeed, the Sac1 protein encoded by the actual Sac1ts allele, TS1, is even more unstable than the change introduced by the original tissue culture experiments in making TS2. These data and their quantification are included in a revised figure (Fig. 1D,E).

Fig. 1.
Sac1 protein is unstable in Sac1ts mutants. (A) Interconversion of PI and PI4P by PI4KII and Sac1. (B) Schematic of Sac1 protein, highlighting conserved domains: Sac domain with conserved motifs (red) and C-terminal transmembrane domains (blue). Sequence alignment of a portion of the Sac domain, including conserved catalytic CX5R(T/S) motif (underlined), the glycine residue mutated in the Sac1ts allele (G382D[TS1]) and in the mutant tested in HeLa and S2 cells (G382R[TS2]) (red), as well as the cysteine residues mutated in Drosophila Sac1 (dSac1) PR (C391S) and human Sac1 (hSac1) PD (C388S) (red). (C) Immunoblots of lysates from HeLa cells expressing different Flag-tagged dSac1 constructs (WT, PR, TS2) at 25°C or 37°C, probed with anti-Flag and anti-GAPDH antibodies (GAPDH, loading control). The shift to non-permissive temperature (37°C) resulted in elevated levels of WT and PR proteins. However, TS2 protein did not accumulate. (D) Representative immunoblot from mock-transfected S2 cells or S2 cells transfected with different HA-tagged dSac1 constructs (WT, G382D[TS1], G382R[TS2]) grown for 3 days at 18°C, then incubated at either 18°C or 29°C for 10 min and probed with anti-HA and anti-GAPDH antibodies (GAPDH, loading control). TS1, the actual mutation encoded by the Sac1ts allele, is even more temperature sensitive in S2 cells than TS2, which was tested in HeLa cells (C; originally designated TS). (E) Densitometry analysis of HA/GAPDH. Results are expressed as arbitrary units. Data points indicate individual values from three independent experiments (n=3), all conducted in S2 cells. The shift to non-permissive temperature (10 min at 29°C) unexpectedly led to elevated levels of the WT protein. Nevertheless, the TS1 and TS2 proteins accumulated at substantially lower levels, consistent with our earlier observation of TS2 in HeLa cells. Asterisks indicate significant differences with respect to HA-Sac1 WT high temp condition (**P<0.01; n.s., not significant, unpaired Student's t-test).
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Sac1 protein is unstable in Sac1ts mutants. (A) Interconversion of PI and PI4P by PI4KII and Sac1. (B) Schematic of Sac1 protein, highlighting conserved domains: Sac domain with conserved motifs (red) and C-terminal transmembrane domains (blue). Sequence alignment of a portion of the Sac domain, including conserved catalytic CX5R(T/S) motif (underlined), the glycine residue mutated in the Sac1ts allele (G382D[TS1]) and in the mutant tested in HeLa and S2 cells (G382R[TS2]) (red), as well as the cysteine residues mutated in Drosophila Sac1 (dSac1) PR (C391S) and human Sac1 (hSac1) PD (C388S) (red). (C) Immunoblots of lysates from HeLa cells expressing different Flag-tagged dSac1 constructs (WT, PR, TS2) at 25°C or 37°C, probed with anti-Flag and anti-GAPDH antibodies (GAPDH, loading control). The shift to non-permissive temperature (37°C) resulted in elevated levels of WT and PR proteins. However, TS2 protein did not accumulate. (D) Representative immunoblot from mock-transfected S2 cells or S2 cells transfected with different HA-tagged dSac1 constructs (WT, G382D[TS1], G382R[TS2]) grown for 3 days at 18°C, then incubated at either 18°C or 29°C for 10 min and probed with anti-HA and anti-GAPDH antibodies (GAPDH, loading control). TS1, the actual mutation encoded by the Sac1ts allele, is even more temperature sensitive in S2 cells than TS2, which was tested in HeLa cells (C; originally designated TS). (E) Densitometry analysis of HA/GAPDH. Results are expressed as arbitrary units. Data points indicate individual values from three independent experiments (n=3), all conducted in S2 cells. The shift to non-permissive temperature (10 min at 29°C) unexpectedly led to elevated levels of the WT protein. Nevertheless, the TS1 and TS2 proteins accumulated at substantially lower levels, consistent with our earlier observation of TS2 in HeLa cells. Asterisks indicate significant differences with respect to HA-Sac1 WT high temp condition (**P<0.01; n.s., not significant, unpaired Student's t-test).

Fig. 1.
Sac1 protein is unstable in Sac1ts mutants. (A) Interconversion of PI and PI4P by PI4KII and Sac1. (B) Schematic of Sac1 protein, highlighting conserved domains: Sac domain with conserved motifs (red) and C-terminal transmembrane domains (blue). Sequence alignment of a portion of the Sac domain, including conserved catalytic CX5R(T/S) motif (underlined), the glycine residue mutated in the Sac1ts allele (G382D[TS1]) and in the mutant tested in HeLa and S2 cells (G382R[TS2]) (red), as well as the cysteine residues mutated in Drosophila Sac1 (dSac1) PR (C391S) and human Sac1 (hSac1) PD (C388S) (red). (C) Immunoblots of lysates from HeLa cells expressing different Flag-tagged dSac1 constructs (WT, PR, TS2) at 25°C or 37°C, probed with anti-Flag and anti-GAPDH antibodies (GAPDH, loading control). The shift to non-permissive temperature (37°C) resulted in elevated levels of WT and PR proteins. However, TS2 protein did not accumulate. (D) Representative immunoblot from mock-transfected S2 cells or S2 cells transfected with different HA-tagged dSac1 constructs (WT, G382D[TS1], G382R[TS2]) grown for 3 days at 18°C, then incubated at either 18°C or 29°C for 10 min and probed with anti-HA and anti-GAPDH antibodies (GAPDH, loading control). TS1, the actual mutation encoded by the Sac1ts allele, is even more temperature sensitive in S2 cells than TS2, which was tested in HeLa cells (C; originally designated TS). (E) Densitometry analysis of HA/GAPDH. Results are expressed as arbitrary units. Data points indicate individual values from three independent experiments (n=3), all conducted in S2 cells. The shift to non-permissive temperature (10 min at 29°C) unexpectedly led to elevated levels of the WT protein. Nevertheless, the TS1 and TS2 proteins accumulated at substantially lower levels, consistent with our earlier observation of TS2 in HeLa cells. Asterisks indicate significant differences with respect to HA-Sac1 WT high temp condition (**P<0.01; n.s., not significant, unpaired Student's t-test).
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Sac1 protein is unstable in Sac1ts mutants. (A) Interconversion of PI and PI4P by PI4KII and Sac1. (B) Schematic of Sac1 protein, highlighting conserved domains: Sac domain with conserved motifs (red) and C-terminal transmembrane domains (blue). Sequence alignment of a portion of the Sac domain, including conserved catalytic CX5R(T/S) motif (underlined), the glycine residue mutated in the Sac1ts allele (G382D[TS1]) and in the mutant tested in HeLa and S2 cells (G382R[TS2]) (red), as well as the cysteine residues mutated in Drosophila Sac1 (dSac1) PR (C391S) and human Sac1 (hSac1) PD (C388S) (red). (C) Immunoblots of lysates from HeLa cells expressing different Flag-tagged dSac1 constructs (WT, PR, TS2) at 25°C or 37°C, probed with anti-Flag and anti-GAPDH antibodies (GAPDH, loading control). The shift to non-permissive temperature (37°C) resulted in elevated levels of WT and PR proteins. However, TS2 protein did not accumulate. (D) Representative immunoblot from mock-transfected S2 cells or S2 cells transfected with different HA-tagged dSac1 constructs (WT, G382D[TS1], G382R[TS2]) grown for 3 days at 18°C, then incubated at either 18°C or 29°C for 10 min and probed with anti-HA and anti-GAPDH antibodies (GAPDH, loading control). TS1, the actual mutation encoded by the Sac1ts allele, is even more temperature sensitive in S2 cells than TS2, which was tested in HeLa cells (C; originally designated TS). (E) Densitometry analysis of HA/GAPDH. Results are expressed as arbitrary units. Data points indicate individual values from three independent experiments (n=3), all conducted in S2 cells. The shift to non-permissive temperature (10 min at 29°C) unexpectedly led to elevated levels of the WT protein. Nevertheless, the TS1 and TS2 proteins accumulated at substantially lower levels, consistent with our earlier observation of TS2 in HeLa cells. Asterisks indicate significant differences with respect to HA-Sac1 WT high temp condition (**P<0.01; n.s., not significant, unpaired Student's t-test).

The designations of these mutations have been revised in the new Fig. 1B. In addition, the designation of the phosphatase-dead (PD) mutant has been corrected from C389S (as published in the original article) to C388S.

The corrected figure and legend are shown below. The article PDF and online full-text version have not been updated.

Yonit Tsatskis (Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada) and Kimberley D. Gauthier (Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada) contributed to the work carried out to correctly characterise the mutation.

The authors apologise to the readers for the errors and any inconvenience they may have caused. The overall findings of the paper are not affected by this error.

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© 2024. Published by The Company of Biologists Ltd
2024