Defining regulatory and phosphoinositide-binding sites in the human WIPI-1 β-propeller responsible for autophagosomal membrane localization downstream of mTORC1 inhibition

Gaugel, Anja; Bakula, Daniela; Hoffmann, Anneliese; Proikas-Cezanne, Tassula

doi:10.1186/1750-2187-7-16

Table 1 Site-directed mutagenesis of human WIPI-1

From: Defining regulatory and phosphoinositide-binding sites in the human WIPI-1 β-propeller responsible for autophagosomal membrane localization downstream of mTORC1 inhibition

Mutant	Position	Mutation	PCR oligonucleotides (forward, reverse)
GFP-N23A	N67-68	AA to GC	AGCTGCTTCTCTTTCGCCCAGGACTGCACATCC,
GFP-N23A	N67-68	AA to GC	GGATGTGCAGTCCTGGGCGAAAGAGAAGCAGCT
GFP-Q24A	N70-71	CA to GC	CTCTTTCAACGCGGACTGCACATCCCTAGCAA,
GFP-Q24A	N70-71	CA to GC	TTGCTAGGGATGTGCAGTCCGCGTTGAAAGAG
GFP-D25A	N74	A to C	TCTTTCAACCAGGCCTGCACATCCCTAGCA,
GFP-D25A	N74	A to C	TGCTAGGGATGTGCAGGCCTGGTTGAAAGA
GFP-E64A	N191	A to C	GTCTACATCGCGGCGCGCCTCTTCTCC,
GFP-E64A	N191	A to C	GGAGAAGAGGCGCGCCGCGATGTAGAC
GFP-R107A	N319-320	AG to GC	CAACATCTTGTCCATAGCGCTGAACCGGCAAAGGC,
GFP-R107A	N319-320	AG to GC	GCCTTTGCCGGTTCAGCGCTATGGACAAGATGTTG
GFP-R110A	N328-329	CG to GC	TCCATAAGGCTGAACGCGCAAAGGCTGCTGGTT,
GFP-R110A	N328-329	CG to GC	AACCAGCAGCCTTTGCGCGTTCAGCCTTATGGA
GFP-R112A	N334-335	AG to GC	GCTGAACCGGCAAGCGCTGCTGGTTTGCC,
GFP-R112A	N334-335	AG to GC	GGCAAACCAGCAGCGCTTGCCGGTTCAGC
GFP-H185A	N553-554	CA to GC	TGCACTATTGCTGCCGCTGAGGGAACACTAGCTGCC,
GFP-H185A	N553-554	CA to GC	GGCAGCTAGTGTTCCCTCAGCGGCAGCAATAGTGCA
GFP-G198A	N593	G to C	CACCTTCAATGCCTCAGCCTCCAAACTAGCA,
GFP-G198A	N593	G to C	TGCTAGTTTGGAGGCTGAGGCATTGAAGGTG
GFP-S203A	N607-608	AG to GC	GGCTCCAAACTAGCAGCTGCGTCTGAAAAAGGC,
GFP-S203A	N607-608	AG to GC	GCCTTTTTCAGACGCAGCTGCTAGTTTGGAGCC
GFP-S205A	N613	T to G	CTAGCAAGTGCGGCTGAAAAAGGCACAGTC,
GFP-S205A	N613	T to G	GACTGTGCCTTTTTCAGCCGCACTTGCTAG
GFP-G208A	N623	G to C	AGTGCGTCTGAAAAAGCCACAGTCATCCGG,
GFP-G208A	N623	G to C	CCGGATGACTGTGGCTTTTTCAGACGCACT
GFP-T209A	N625	A to G	TCTGAAAAAGGCGCAGTCATCCGGGTG,
GFP-T209A	N625	A to G	CACCCGGATGACTGCGCCTTTTTCAGA
GFP-R212A	N634-635	CG to GC	GGCACAGTCATCGCGGTGTTCTCTGTCCC,
GFP-R212A	N634-635	CG to GC	GGGACAGAGAACACCGCGATGACTGTGCC
GFP-E224A	N671	A to C	GGCAAAAGCTCTATGCGTTCCGGAGAGG,
GFP-E224A	N671	A to C	CCTCTCCGGAACGCATAGAGCTTTTGCC
GFP-F225A	N673-674	TT to GC	GCAAAAGCTCTATGAGGCCCGGAGAGGGATGAA,
GFP-F225A	N673-674	TT to GC	TTCATCCCTCTCCGGGCCTCATAGAGCTTTTGC
GFP-R226A	N676-677	CG to GC	CAAAAGCTCTATGAGTTCGCGAGAGGGATGAAAAGGTATG,
GFP-R226A	N676-677	CG to GC	CATACCTTTTCATCCCTCTCGCGAACTCATAGAGCTTTTG
GFP-R227A	N679-680	AG to GC	AAAAGCTCTATGAGTTCCGGGCAGGGATGAAAAGGTATGT,
GFP-R227A	N679-680	AG to GC	ACATACCTTTTCATCCCTGCCCGGAACTCATAGAGCTTTT
GFP-RR	N676-677	CG to GC	GGGCAAAAGCTCTATGAGTTCGCTGCAGGGATGAAAAGGTATGTGAC,
GFP-RR	N679-680	AG to GC	GTCACATACCTTTTCATCCCTGCAGCGAACTCATAGAGCTTTTGCCC
GFP-G228A	N683	G to C	CTATGAGTTCCGGAGAGCGATGAAAAGGTATGTG,
GFP-G228A	N683	G to C	CACATACCTTTTCATCGCTCTCCGGAACTCATAG
GFP-S250A	N748	T to G	CCTCTGCGCCGCCAGTAACACCGAG,
GFP-S250A	N748	T to G	CTCGGTGTTACTGGCGGCGCAGAGG
GFP-S251A	N751-752	AG to GC	CTCTGCGCCTCCGCTAACACCGAGACG,
GFP-S251A	N751-752	AG to GC	CGTCTCGGTGTTAGCGGAGGCGCAGAG
GFP-T255A	N763	A to G	CCAGTAACACCGAGGCGGTACACATCTTC,
GFP-T255A	N763	A to G	GAAGATGTGTACCGCCTCGGTGTTACTGG
GFP-H257A	N769-770	CA to GC	CACCGAGACGGTAGCCATCTTCAAGCTGGAAC,
GFP-H257A	N769-770	CA to GC	GTTCCAGCTTGAAGATGGCTACCGTCTCGGTG
GFP-S335A	N1003-1004	AG to GC	GCTAGTTGCGTCATCCGCTGGACACCTTTATATG,
GFP-S335A	N1003-1004	AG to GC	CATATAAAGGTGTCCAGCGGATGACGCAACTAGC
GFP-G336A	N1007	G to C	CTAGTTGCGTCATCCAGTGCACACCTTTATATG,
			CATATAAAGGTGTGCACTGGATGACGCAACTAG

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