Structural Conservation of the Two Phosphoinositide-Binding Sites in WIPI Proteins

Liang, Rongrui; Ren, Jinqi; Zhang, Yong; Feng, Wei

doi:10.1016/j.jmb.2019.02.019

Cited by 33 publications

(50 citation statements)

References 39 publications

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“…For purification in E.coli, a C-terminal 1D4 epitope tag (TETSQVAPA) was added to 2xHrs by PCR, then sub-cloned into the pGEX vector (GE Life Sciences), thereby also adding an N-terminal GST tag. Residues in the 4-amino acid phosphoinositide binding sequence FRRG in mouse WIPI2 were mutated to FTRG or FTTG (Baskaran et al, 2012;Liang et al, 2019) by PCR site-directed mutagenesis. WT mouse WIPI2, WIPI2 FTRG , and WIPI2 FTTG with N-terminal GST fusions and C-terminal 1D4 tags were cloned into BamHI and NotI sites in pFb1 vector (Thermo).…”

Section: Methodsmentioning

confidence: 99%

“…WIPI2 is a member of the PROPPIN family, which is well known for binding to both PI(3)P and PI(3,5)P2 (Dove et al, 2004(Dove et al, , 2009Liang et al, 2019), and human WIPI2 was reported to bind PI(3)P, PI(3,5)P2, PI(4)P, and PI(5)P in lipid overlay assays (Liang et al, 2019;Vicinanza et al, 2015). However, we found that mouse GST-WIPI2 phosphoinositide binding was highly specific for PI(3)P, although binding was weak compared to GST-2xHrs ( Fig 7A).…”

Section: Lc3-positive Autophagosomes Accumulate In Knockout Rpementioning

confidence: 99%

“…In titrations of PI(3)P in PI-ELISA assays (He et al, 2016) GST-WIPI2 had a ~6-fold lower affinity for PI(3)P than GST-2xHrs (K1/2 values were 2.2 x 10 -3 (±1.8 x10 -4 ) and 3.8 x 10 -4 (±3.9 x 10 -5 )(SEM, n=3) mol PI(3)P/mol total phospholipid, for WIPI2 and 2xHrs, respectively; corresponding to surface densities of roughly 494 or 2800 molecules/µm 2 , assuming a nominal area per phospholipid of 77 A 2 (Burke et al, 1973) (Fig 7C). WIPI2 has two PI binding sites coordinated by the conserved FRRG motif (Baskaran et al, 2012;Krick et al, 2012;Liang et al, 2019).…”

Section: Lc3-positive Autophagosomes Accumulate In Knockout Rpementioning

confidence: 99%

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Multiple phosphatidylinositol(3)phosphate roles in retinal pigment epithelium membrane recycling

et al. 2020

Preprint

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The low-abundance lipid phosphatidylinositol-3-phosphate (PI(3)P) is important for membrane dynamics in autophagy, endosome processing, and phagocytosis. In retinal pigmented epithelial cells (RPE) all three pathways are important, but phagocytosis of disk membranes shed from adjacent photoreceptors is especially important for ensuring health of photoreceptors and preventing retinal degeneration. By eliminating Vps34, the kinase responsible for synthesizing PI(3)P in RPE, we have found that PI(3)P plays distinct roles in each pathway. In phagocytosis it is not required for disk engulfment or phagosome transport but is essential for recruitment and lipidation of LC3. In contrast, initiation of autophagy and LC3 recruitment to autophagosomes does not require PI(3)P, which can be bypassed by an alternative mechanism of ATG16L recruitment that does not require PI(3)P, Rab11a, or WIPI2. In all three pathways, PI(3)P is essential for fusion with lysosomes; autophagosomes, phagosomes, and Rab7-positive late endosomes, as well as enlarged lysosomes, accumulate in large numbers in the absence of Vps34, leading to cell death.

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Section: Methodsmentioning

confidence: 99%

Section: Lc3-positive Autophagosomes Accumulate In Knockout Rpementioning

confidence: 99%

Section: Lc3-positive Autophagosomes Accumulate In Knockout Rpementioning

confidence: 99%

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Multiple phosphatidylinositol(3)phosphate roles in retinal pigment epithelium membrane recycling

et al. 2020

Preprint

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“…The C‐terminal half appears to go back to the N terminus, thereby producing an antiparallel topology, although the precise location of the C terminus of ATG2 in the rod structure is unclear due to its flexible conformation. Atg18 homologs belong to the PROPPIN family, which has a seven‐bladed β‐propeller fold with two phosphoinositide‐binding pockets that have been clearly characterized using high‐resolution crystal structures of Atg18 homologs . The β‐propeller ring of WIPI4 binds to one end of the ATG2 rod.…”

Section: Shape Of the Atg2–atg18 Complexmentioning

confidence: 99%

“…Atg18 homologs belong to the PROPPIN family, which has a seven-bladed β-propeller fold with two phosphoinositide-binding pockets that have been clearly characterized using highresolution crystal structures of Atg18 homologs. [31][32][33][34][35] The β-propeller ring of WIPI4 binds to one end of the ATG2 rod. Thus, the overall shape of the ATG2-WIPI4 complex looks like a golf club.…”

Section: Shape Of the Atg2-atg18 Complexmentioning

confidence: 99%

Atg2: A novel phospholipid transfer protein that mediates de novo autophagosome biogenesis

Osawa

Noda

2019

Protein Science

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The degradation of cytoplasmic components via autophagy is crucial for intracellular homeostasis. In the process of autophagy, a newly synthesized isolation membrane (IM) is developed to sequester degradation targets and eventually the IM seals, forming an autophagosome. One of the most poorly understood autophagy‐related proteins is Atg2, which is known to localize to a contact site between the edge of the expanding IM and the exit site of the endoplasmic reticulum (ERES). Recent advances in structural and biochemical analyses have been applied to Atg2 and have revealed it to be a novel multifunctional protein that tethers membranes and transfers phospholipids between them. Considering that Atg2 is essential for the expansion of the IM that requires phospholipids as building blocks, it is suggested that Atg2 transfers phospholipids from the ERES to the IM during the process of autophagosome formation, suggesting that lipid transfer proteins can mediate de novo organelle biogenesis.

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Functional evidence for a de novo mutation in WDR45 leading to BPAN in a Chinese girl

Xiong

et al. 2019

Molec Gen & Gen Med

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Background Beta‐propeller protein‐associated neurodegeneration (BPAN, OMIM 300894) is an X‐linked neurodegenerative disorder caused by mutations in WDR45. WDR45 is required for autophagy, defect in WDR45 impaired autophagy which contributes for the pathogenesis of BPAN. Previously, we reported a novel de novo mutation (c.1040_1041del, p.Glu347GlyfsTer7) in WDR45 (NM_007075) in a 3‐year‐old Chinese girl with BPAN. Methods The protein structure was constructed using SWISS‐MODEL and the isoelectric point (pI) was predicted by the online pI/Mw tool at ExPASy. The functional effects of this mutation were predicted by two online software programs: PROVEN and MutationTaster. Stable overexpression of Flag‐tagged wild‐type or mutant WDR45 in HeLa cells was constructed. Protein levels of LC3 and p62 were analyzed by western blot upon treatment with/without autophagy inhibitor Bafilomycin A1, the formation of LC3 puncta were analyzed in HeLa cells transfected with mCherry‐LC3 by confocal microscopy. Results The mutation resulted in a shift of pI from 6.74 to 8.84 and was predicted to be pathogenic. The protein levels of LC3‐II and p62 were increased in cells overexpression of wild‐type and mutant WDR45 while the protein levels were not increased in cells overexpression of mutant WDR45 upon treatment with autophagy inhibitor Bafilomycin A1. Results from confocal microscopy revealed that LC3‐positive puncta were increased in cells expressing both wild‐type and mutant WDR45 while the number of LC3‐positive puncta was not increased in cells expressing mutant WDR45 upon treatment with Bafilomycin A1. Conclusion Our study evidenced that this novel mutation in WDR45 impaired autophagy in cells thus this mutation is the cause for BPAN in this patient.

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Structural Conservation of the Two Phosphoinositide-Binding Sites in WIPI Proteins

Cited by 33 publications

References 39 publications

Multiple phosphatidylinositol(3)phosphate roles in retinal pigment epithelium membrane recycling

Multiple phosphatidylinositol(3)phosphate roles in retinal pigment epithelium membrane recycling

Atg2: A novel phospholipid transfer protein that mediates de novo autophagosome biogenesis

Functional evidence for a de novo mutation in WDR45 leading to BPAN in a Chinese girl

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