Elevating PI3P drives select downstream membrane trafficking pathways

Abstract

Phosphoinositide signaling lipids are essential for several cellular processes. The requirement for a phosphoinositide is conventionally studied by depleting the corresponding lipid kinase. However, there are very few reports on the impact of elevating phosphoinositides. That phosphoinositides are dynamically elevated in response to stimuli suggests that, in addition to being required, phosphoinositides drive downstream pathways. To test this hypothesis, we elevated the levels of phosphatidylinositol-3-phosphate (PI3P) by generating hyperactive alleles of the yeast phosphatidylinositol 3-kinase, Vps34. We find that hyperactive Vps34 drives certain pathways, including PI(3,5)P2 synthesis and retrograde transport from the vacuole. This demonstrates that PI3P is rate limiting in some pathways. Interestingly, hyperactive Vps34 does not affect ESCRT function. Thus, elevating PI3P does not always increase the rate of PI3P-dependent pathways. Elevating PI3P can also delay a pathway. Elevating PI3P slowed late steps in autophagy, in part by delaying the disassembly of autophagy proteins from mature autophagosomes as well as delaying fusion of autophagosomes with the vacuole. This latter defect is likely due to a more general defect in vacuole fusion, as assessed by changes in vacuole morphology. These studies suggest that stimulus-induced elevation of phosphoinositides provides a way for these stimuli to selectively regulate downstream processes.

Activation Mechanisms of the VPS34 Complexes

“…No dislodging effect of the HELCAT was found in this study, and the authors speculated that dislodging is an intermediate state [48]. Using the autoinhibition information, Steinfeld et al designed active mutants for the yeast Vps34-Vps15 heterodimer by introducing point mutations at an interface between the Vps34 HELCAT and Vps15 kinase domain [54]. Unlike in human complex I, the kinase domains of Vps34 and Vps15 are tightly packed in the yeast Vps34-Vps15 heterodimer [55].…”

Endosomal cargo recycling mediated by Gpa1 and Phosphatidylinositol-3-Kinase is inhibited by glucose starvation

“…Both vps15Δ and vps34Δ mutants were confirmed to have a growth defect at 30°C ( Figure 4B ) before we revealed that vps15Δ and vps34Δ cells are severely defective in their ability to recycle internalised FM4-64 dye ( Figure 4C ). To further corroborate this model, we employed a hyperactive version of Vps34, termed Vps34 EDC (harbouring R283E, A287D, Y501C point mutations) that was recently used to show PtdIns3P production is rate limiting for some, but not all, membrane trafficking pathways (Steinfeld et al ., 2021). We found that unlike deletion of PI3K, increased expression of Vps34 WT or Vps34 EDC had no effect on growth at 30°C ( Figure 4B ).…”

“…Instead, we performed experiments with Mat A vps15∆ and vps34∆ mutants, that are both extremely defective in FM4-64 recycling. To further test the role of PI3K in recycling, we took advantage of an optimised hyperactive Vps34 allele that stimulates over-production of PtdIns3P, which had previously been shown to upregulate retrograde trafficking, perturb late stages of autophagy and have no effect of MVB sorting (Steinfeld et al, 2021). Expression of hyperactive Vps34 resulted in defects in FM4-64 recycling (Figure 3F) suggesting, like the late stages of autophagy, recycling to the surface requires specific PtdIns3P regulation.…”

Deliver on Time or Pay the Fine: Scheduling in Membrane Trafficking

“…Stability of the Retromer complex to the newborn vesicle strictly depends on SNX proteins, as consistent SNXs depletion results in a block of the sorting process [81]. Similarly, reduction of PtdIns(3)P, the SNXs regulator, results in a similar phenotype [143,145].…”