Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors

Abstract

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), made by Fab1p, is essential for vesicle recycling from vacuole/lysosomal compartments and for protein sorting into multivesicular bodies. To isolate PtdIns(3,5)P2 effectors, we identified Saccharomyces cerevisiae mutants that display fab1delta-like vacuole enlargement, one of which lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p displays PtdIns(3,5)P2 binding of exquisite specificity, GFP-Svp1p localises to the vacuole membrane in a Fab1p-dependent manner, and svp1delta cells fail to recycle a marker protein from the vacuole to the Golgi. Cells lacking Svp1p accumulate abnormally large amounts of PtdIns(3,5)P2. These observations identify Svp1p as a PtdIns(3,5)P2 effector required for PtdIns(3,5)P2-dependent membrane recycling from the vacuole. Other Svp1p-related proteins, including human and Drosophila homologues, bind PtdIns(3,5)P2 similarly. Svp1p and related proteins almost certainly fold as beta-propellers, and the PtdIns(3,5)P2-binding site is on the beta-propeller. It is likely that many of the Svp1p-related proteins that are ubiquitous throughout the eukaryotes are PtdIns(3,5)P2 effectors. Svp1p is not involved in the contributions of FAB1/PtdIns(3,5)P2 to MVB sorting or to vacuole acidification and so additional PtdIns(3,5)P2 effectors must exist.

Determining selectivity of phosphoinositide-binding domains

“…However, when PH or FYVE domain binding to phosphoinositides is studied, there appears to be more of a discrepancy. Monitoring steady state RU signals, we achieve excellent agreement in K D values (0.6-3 µM) for PtdIns(4,5)P 2 binding by the PLC 1 PH domain between our SPR studies [56,57], ITC [39], and centrifugation approaches [20]. Similarly, estimates for PtdIns3P binding by the Hrs1 FYVE domain are within 2-fold when comparing results from SPR (K D = 4.8 µM [57]) and centrifugation assays (K D = 2.5 µM [46]).…”

“…Monitoring steady state RU signals, we achieve excellent agreement in K D values (0.6-3 µM) for PtdIns(4,5)P 2 binding by the PLC 1 PH domain between our SPR studies [56,57], ITC [39], and centrifugation approaches [20]. Similarly, estimates for PtdIns3P binding by the Hrs1 FYVE domain are within 2-fold when comparing results from SPR (K D = 4.8 µM [57]) and centrifugation assays (K D = 2.5 µM [46]). By contrast, using k off /k on ratios, Cho and colleagues report a 100-fold higher apparent affinity for the Hrs1/PtdIns3P interaction [58] than we have measured, the origin (or validity) of which is not clear.…”

“…As mentioned previously, the reduced water solubility of PtdIns3P/PtdIns4P/ PtdIns5P causes the apparent binding to these phosphoinositides to be over-represented in dot-blot/lipid overlay studies compared with binding to PtdInsP 2 isomers including PtdIns(3,5)P 2 . We suggest that this provides the explanation for the discrepancy between the findings of Strømhaug et al [70] and those presented in Figure 3B [57]. According to the methodological considerations presented in this article, we are confident that yeast Atg18p, Atg21p and Hsv2p/Ygr223cp are specific for PtdIns(3,5)P 2 under physiological conditions.…”

“…By contrast with these examples, a recent study by Dove et al [57], including the SPR approach described here, identified the S. cerevisiae protein Atg18p (also called Svp1p) as a highly specific PtdIns(3,5)P 2 -binding protein. The clear specificity of Atg18p for PtdIns(3,5)P 2 over PtdIns(4,5)P 2 and PtdIns3P is illustrated in the SPR data shown in Figure 3B.…”

“…A related Drosophila protein, known as Dm3, shows a similar specificity for PtdIns(3,5)P 2 over PtdIns(4,5)P 2 in this assay, but does not appear to distinguish PtdIns(3,5)P 2 from PtdIns3P, which has significant functional implications. Atg18p/Svp1p was first identified as a possible PtdIns(3,5)P 2 effector on the basis that its deletion in S. cerevisiae gives rise to a phenotype similar to that seen when yeast do not express Fab1p, the PtdIns3P 5-kinase that produces PtdIns(3,5)P 2 [57]. The PtdIns(3,5)P 2 specificity seen for Atg18p/Svp1p in our vesicle studies was also recapitulated in studies using pure phosphoinositides, although the dot-blot/lipid overlay approach appears to have greatly overestimated the extent to which it binds PtdIns3P -possibly as a result of the lipid solubility issue mentioned in Section 2.2.3.…”