The Fab1 phosphatidylinositol kinase pathway in the regulation of vacuole morphology

doi:10.1016/j.ceb.2005.06.002

Current Opinion in Cell Biology

Volume 17, Issue 4, August 2005, Pages 402-408

https://doi.org/10.1016/j.ceb.2005.06.002 Get rights and content

Yeast vacuoles are very dynamic structures that must respond to changes in extracellular osmolarity by rapidly altering their size, thereby releasing or taking up water and ions. Further, the need to accommodate a constant biosynthetic influx of membrane and to partition vacuoles during cell division necessitates precise regulation of the size and shape of the vacuole. While it is has been shown that the lipid kinase Fab1p and its product phosphatidylinositol 3,5-bisphosphate, and not the mitogen-activated protein kinase Hog1p, are central to this regulatory pathway, key effectors still await identification. Atg18p is the most recently identified candidate for a Fab1p effector mediating the largely uncharacterized processes of vesicle fission and membrane recycling at the vacuole.

Introduction

The Saccharomyces cerevisiae vacuole, the yeast analog of the mammalian lysosome, is a highly versatile organelle. It serves as the main storage compartment for essential amino acids, nutrients and ions, functions in the turnover of macromolecules, and is required for sporulation and osmotic homeostasis [1]. Not surprisingly, the vacuole has evolved to be very dynamic; rapid changes in size, shape and number are critical to ensure an immediate response to dramatic fluctuations in extracellular osmolarity and nutrient concentrations.

However, even under relatively static external conditions, the vacuole is in a constant state of regulated flux. For example, the cell-cycle-dependent process of vacuole inheritance involves significant changes in vacuole morphology and requires precise regulation [2]. In addition, the multiple endocytic and biosynthetic membrane transport pathways that converge on the vacuole as well as pathways for recycling and degrading membrane constituents (i.e. lipids and proteins) are kept in balance to maintain the proper size and shape of the vacuole [3, 4].

To date, a complete picture of how yeast regulate vacuole membrane dynamics has not yet emerged. In terms of biogenesis, the core fusion machinery of biosynthetic sorting pathways — comprising Vam3p (a t-SNARE), the class C vacuolar protein sorting/HOPS complex (which functions in tethering) and Ytp7p (a Rab GTPase) — plays a critical role in fusion at the vacuole [5, 6, 7, 8]. Additionally, actin, phosphoinositides, ergosterol, diacylglycerol, V-ATPase activity and ion regulation also affect fusion [9, 10, 11, 12]. Nonetheless, the impact these fusion and biogenesis factors have on vacuole size regulation is only part of the story.

Over the past decade, a plethora of studies have implicated phosphoinositides as key determinants of organelle identity and morphology, and the vacuole is no exception: a growing body of genetic and biochemical evidence suggests that the regulated synthesis and turnover of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) underlies many aspects of vacuole function [2, 13].

This review examines various facets of vacuole size control in yeast, with an emphasis on the potential roles of the kinase Fab1p and its product PtdIns(3,5)P₂.

Section snippets

PtdIns(3,5)P₂ and vacuole size regulation

PtdIns(3,5)P₂ on the vacuole is generated by Fab1p (the mammalian homolog is called PIKfyve), the sole phosphatidylinositol 3-phosphate (PtdIns3P) 5-kinase in yeast. As a substrate, Fab1p utilizes PtdIns3P generated by the PtdIns 3-kinase Vps34p [14, 15, 16]. Fab1p is intimately involved in vacuole size regulation, presumably by modulating PtdIns(3,5)P₂ effector activity [17^••].

A fab1Δ strain entirely devoid of PtdIns(3,5)P₂ displays a complex, pleiotropic phenotype consisting of a dramatically

Osmoregulation: separate roles for Hog1p and Fab1p

To achieve long-term adaptation to hypo- or hyperosmotic stress, S. cerevisiae uses the protein kinase C (cell integrity) and HOG-MAP (high osmolarity glycerol mitogen-actived protein) kinase pathways, respectively [38]. It has recently been shown that phosphorylation (and subsequent activation) of plasma membrane channels such as NHA1 (Na⁺/H⁺ antiporter) by Hog1p plays a critical role in the immediate response to osmotic shock [39]. Rapid changes in vacuole size and volume (resulting from

Vacuolar inheritance

Throughout the cell cycle, yeast must transfer their vacuoles in a regulated fashion to ensure proper inheritance by daughter cells. In budding yeast, vacuolar membrane is rapidly transported into a nascent bud via a segregation structure. This membraneous projection mediates traffic — potentially vesicular — between mother cell and the newly formed vacuole [41]. This myosin-mediated exchange of membrane continues along actin tracks until late in the cell division cycle [42, 43]. Accordingly,

Conclusions

The processes of anterograde and retrograde membrane trafficking, osmoregulation and vacuole inheritance are all linked to vacuole size regulation. While the major pathways governing these processes have been identified, a complete picture has yet to emerge. However, the recent identification of Atg18p as an effector of PtdIns(3,5)P₂ constitutes a very important step forward. Undoubtedly, identifying other novel effectors will be critical in solving the complex regulation of PtdIns(3,5)P₂

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

We would like to thank Simon Rudge, William Parrish, and Chris Stefan for helpful discussions and critical reading of the manuscript. RJB is supported by the Canadian Institutes for Health Research, and SDE is an investigator of the Howard Hughes Medical Institute.

References (47)

G. Odorizzi et al.
Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body
Cell
(1998)
T.K. Sato et al.
Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion
Mol Cell
(2000)
Y. Jun et al.
Diacylglycerol and its formation by phospholipase C regulate Rab- and SNARE-dependent yeast vacuole fusion
J Biol Chem
(2004)
O.C. Ikonomov et al.
Mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 5-kinase PIKfyve
J Biol Chem
(2001)
C.J. Bonangelino et al.
Vac7p, a novel vacuolar protein, is required for normal vacuole inheritance and morphology
Mol Cell Biol
(1997)
C. Peters et al.
Mutual control of membrane fission and fusion proteins
Cell
(2004)
D.J. Katzmann et al.
Receptor downregulation and multivesicular-body sorting
Nat Rev Mol Cell Biol
(2002)
O. Muller et al.
Autophagic tubes: vacuolar invaginations involved in lateral membrane sorting and inverse vesicle budding
J Cell Biol
(2000)
M. Proft et al.
MAP kinase-mediated stress relief that precedes and regulates the timing of transcriptional induction
Cell
(2004)
M. Latterich et al.
Evidence for a dual osmoregulatory mechanism in the yeast Saccharomyces cerevisiae
Biochem Biophys Res Commun
(1993)

F. Tang et al.

Regulated degradation of a class V myosin receptor directs movement of the yeast vacuole

Nature

(2003)

B.K. Han et al.

The G1 cyclin Cln3p controls vacuolar biogenesis in Saccharomyces cerevisiae

Genetics

(2003)

N.J. Bryant et al.

Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuole

Microbiol Mol Biol Rev

(1998)

L.S. Weisman

Yeast vacuole inheritance and dynamics

Annu Rev Genet

(2003)

N.J. Bryant et al.

Retrograde traffic out of the yeast vacuole to the TGN occurs via the prevacuolar/endosomal compartment

J Cell Biol

(1998)

A.E. Wurmser et al.

New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion

J Cell Biol

(2000)

A. Haas et al.

The GTPase Ypt7p of Saccharomyces cerevisiae is required on both partner vacuoles for the homotypic fusion step of vacuole inheritance

EMBO J

(1995)

M.R. Peterson et al.

The class C Vps complex functions at multiple stages of the vacuolar transport pathway

Traffic

(2001)

V.J. Starai et al.

Ion regulation of homotypic vacuole fusion in Saccharomyces cerevisiae

J Biol Chem

(2005)

R.A. Fratti et al.

Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles

J Cell Biol

(2004)

W. Wickner

Yeast vacuoles and membrane fusion pathways

EMBO J

(2002)

K. Peplowska et al.

Expanding dynamin: from fission to fusion

Nat Cell Biol

(2005)

J.D. Gary et al.

Fab1p is essential for PtdIns(3)P 5-kinase activity and the maintenance of vacuolar size and membrane homeostasis

J Cell Biol

(1998)

Cited by (81)

PIP kinases: A versatile family that demands further therapeutic attention
2023, Advances in Biological Regulation
Conventional and Secretory Lysosomes
2022, Encyclopedia of Cell Biology: Volume 1-6, Second Edition
Lysosomes are membrane-bound organelles that contain hydrolases capable of degrading proteins, lipids, and carbohydrates. They are involved in nutrient sensing and storage and retrieval. Lysosomes are highly dynamic and are capable of fusion and fission events with other organelles and plasma membrane. Higher eukaryotes possess a specialized group of lysosomes termed secretory lysosomes that are involved in pigmentation, coagulation, wound repair, and immunologic functions. Understanding the molecular machinery that regulates lysosome and secretory lysosome homeostasis is underscored by the fact that mutations in the genes that regulate the biogenesis, movement and delivery of lysosome and secretory lysosome contents result in human diseases.
Lysosomal Ca2+ release channel TRPML1 regulates lysosome size by promoting mTORC1 activity
2019, European Journal of Cell Biology
Citation Excerpt :
Therefore, inhibiting CaM suppresses lysosome fission whereas lysosome fusion is not largely affected, leading to enlarged lysosomes (Fig. 5A). Supporting our hypothesis, an increase in PI(3,5)P2, an endogenous activator of TRPML1 (Dong et al., 2010a), has also been associated with the fission of yeast vacuoles, the counterpart of mammalian lysosomes (Rudge et al., 2004; Efe et al., 2005). Deficiency in PI(3,5)P2 leads to defects in the lysosome fission (Zou et al., 2015), lysosomal reformation (Bissig et al., 2017) and enlarged lysosomes (Cheng et al., 2010; Dong et al., 2010a; Bissig et al., 2017; Choy et al., 2018), and activating TRPML1 rescues the defects in PI(3,5)P2 deficient cells (Zou et al., 2015).
Lysosomal Ca²⁺ release channel TRPML1 has been suggested to regulate lysosome size by activating calmodulin (CaM). To further understand how TRPML1 and CaM regulate lysosome size, in this study, we report that inhibiting mTORC1 causes enlarged lysosomes, and the recovery of enlarged lysosomes is suppressed by inhibiting mTORC1. We also show that lysosome vacuolation induced by inhibiting TRPML1 is corrected by mTORC1 upregulation, and the facilitating effect of TRPML1 on the recovery of enlarged lysosomes is suppressed by inhibiting mTORC1. In the meantime, lysosome vacuolation induced by inhibiting CaM is corrected by mTORC1 upregulation, and mTORC1 overexpression corrects the inhibitory effect of CaM antagonist on the recovery of enlarged lysosomes. Conversely, the vacuolation induced by suppressing mTORC1 is not corrected by upregulating CaM. These data suggest that mTORC1 functions downstream of TRPML1 and CaM to regulate lysosome size. Together with our recent finding showing that TRPML1, CaM and mTORC1 form a macromolecular complex to control mTORC1 activity, we suggest that TRPML1 and CaM control lysosome fission through regulating mTORC1, identifying an mTORC1-dependent molecular mechanism for lysosomal membrane fission.
Phosphatidylinositol 3,5-bisphosphate is involved in methylglyoxal-induced activation of the Mpk1 mitogen-activated protein kinase cascade in Saccharomyces cerevisiae
2017, Journal of Biological Chemistry
Citation Excerpt :
In the present study, we focused on the physiological role of PtdIns(3,5)P2 with respect to MG-induced stress responses. PtdIns(3,5)P2 is enriched in the vacuolar membrane and is involved in the control of vacuolar morphology and functions (22, 30–34). Previous studies reported that mutants defective in the production of PtdIns(3,5)P2, i.e. vps34Δ, vps15Δ, fab1Δ, vac7Δ, and vac14Δ, have a single grossly enlarged vacuole (21, 22, 35, 36).
Methylglyoxal (MG) is a natural metabolite derived from glycolysis, and this 2-oxoaldehyde has been implicated in some diseases including diabetes. However, the physiological significance of MG for cellular functions is yet to be fully elucidated. We previously reported that MG activates the Mpk1 (MAPK) cascade in the yeast Saccharomyces cerevisiae. To gain further insights into the cellular functions and responses to MG, we herein screened yeast-deletion mutant collections for susceptibility to MG. We found that mutants defective in the synthesis of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) are more susceptible to MG. PtdIns(3,5)P₂ levels increased following MG treatment, and vacuolar morphology concomitantly changed to a single swollen shape. MG activated the Pkc1–Mpk1 MAPK cascade in which a small GTPase Rho1 plays a crucial role, and the MG-induced phosphorylation of Mpk1 was impaired in mutants defective in the PtdIns(3,5)P₂ biosynthetic pathway. Of note, heat shock-induced stress also provoked Mpk1 phosphorylation in a Rho1-dependent manner; however, PtdIns(3,5)P₂ was dispensable for the heat shock-stimulated activation of this signaling pathway. Our results suggest that PtdIns(3,5)P₂ is specifically involved in the MG-induced activation of the Mpk1 MAPK cascade and in the cellular adaptation to MG-induced stress.
The m6A methyltransferase Ime4 epitranscriptionally regulates triacylglycerol metabolism and vacuolar morphology in haploid yeast cells
2017, Journal of Biological Chemistry
N⁶-Methyladenosine (m⁶A) is among the most common modifications in eukaryotic mRNA. The role of yeast m⁶A methyltransferase, Ime4, in meiosis and sporulation in diploid strains is very well studied, but its role in haploid strains has remained unknown. Here, with the help of an immunoblotting strategy and Ime4-GFP protein localization studies, we establish the physiological role of Ime4 in haploid cells. Our data showed that Ime4 epitranscriptionally regulates triacylglycerol metabolism and vacuolar morphology through the long-chain fatty acyl-CoA synthetase Faa1, independently of the RNA methylation complex (MIS complex). The MIS complex consists of the Ime4, Mum2, and Slz1 proteins. Our affinity enrichment strategy (methylated RNA immunoprecipitation assays) using m⁶A polyclonal antibodies coupled with mRNA isolation, quantitative real-time PCR, and standard PCR analyses confirmed the presence of m⁶A-modified FAA1 transcripts in haploid yeast cells. The term “epitranscriptional regulation” encompasses the RNA modification-mediated regulation of genes. Moreover, we demonstrate that the Aft2 transcription factor up-regulates FAA1 expression. Because the m⁶A methylation machinery is fundamentally conserved throughout eukaryotes, our findings will help advance the rapidly emerging field of RNA epitranscriptomics. The metabolic link identified here between m⁶A methylation and triacylglycerol metabolism via the Ime4 protein provides new insights into lipid metabolism and the pathophysiology of lipid-related metabolic disorders, such as obesity. Because the yeast vacuole is an analogue of the mammalian lysosome, our findings pave the way to better understand the role of m⁶A methylation in lysosome-related functions and diseases.
The lysosomal Ca2+ release channel TRPML1 regulates lysosome size by activating calmodulin
2017, Journal of Biological Chemistry
Citation Excerpt :
Our studies suggest that TRPML1 may facilitate lysosomal membrane fission through a CaM-dependent mechanism. Because cells with deficiency in either TRPML1 or PI3,5P2, the endogenous TRPML1 agonist, display enlarged lysosomes (10, 14) and because PI3,5P2 has been associated with the fission of yeast vacuole, the counterpart of mammalian lysosome (29, 33, 34), we hypothesized that TRPML1 may control lysosome fission. To test this, we first treated Cos1 cells with vacuolin-1, a chemical that enlarges lysosomes (15, 35).
Intracellular lysosomal membrane trafficking, including fusion and fission, is crucial for cellular homeostasis and normal cell function. Both fusion and fission of lysosomal membrane are accompanied by lysosomal Ca²⁺ release. We recently have demonstrated that the lysosomal Ca²⁺ release channel P2X4 regulates lysosome fusion through a calmodulin (CaM)-dependent mechanism. However, the molecular mechanism underlying lysosome fission remains uncertain. In this study, we report that enlarged lysosomes/vacuoles induced by either vacuolin-1 or P2X4 activation are suppressed by up-regulating the lysosomal Ca²⁺ release channel transient receptor potential mucolipin 1 (TRPML1) but not the lysosomal Na⁺ release channel two-pore channel 2 (TPC2). Activation of TRPML1 facilitated the recovery of enlarged lysosomes/vacuoles. Moreover, the effects of TRPML1 on lysosome/vacuole size regulation were eliminated by Ca²⁺ chelation, suggesting a requirement for TRPML1-mediated Ca²⁺ release. We further demonstrate that the prototypical Ca²⁺ sensor CaM is required for the regulation of lysosome/vacuole size by TRPML1, suggesting that TRPML1 may promote lysosome fission by activating CaM. Given that lysosome fission is implicated in both lysosome biogenesis and reformation, our findings suggest that TRPML1 may function as a key lysosomal Ca²⁺ channel controlling both lysosome biogenesis and reformation.

View all citing articles on Scopus

View full text

The Fab1 phosphatidylinositol kinase pathway in the regulation of vacuole morphology

Introduction

Section snippets

PtdIns(3,5)P2 and vacuole size regulation

Osmoregulation: separate roles for Hog1p and Fab1p

Vacuolar inheritance

Conclusions

References and recommended reading

Acknowledgements

Cell

Mol Cell

J Biol Chem

J Biol Chem

Mol Cell Biol

Cell

Nat Rev Mol Cell Biol

J Cell Biol

Cell

Biochem Biophys Res Commun

Nature

Genetics

Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuole

Microbiol Mol Biol Rev

Yeast vacuole inheritance and dynamics

Annu Rev Genet

Retrograde traffic out of the yeast vacuole to the TGN occurs via the prevacuolar/endosomal compartment

J Cell Biol

New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion

J Cell Biol

The GTPase Ypt7p of Saccharomyces cerevisiae is required on both partner vacuoles for the homotypic fusion step of vacuole inheritance

EMBO J

The class C Vps complex functions at multiple stages of the vacuolar transport pathway

Traffic

Ion regulation of homotypic vacuole fusion in Saccharomyces cerevisiae

J Biol Chem

Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles

J Cell Biol

Yeast vacuoles and membrane fusion pathways

EMBO J

Expanding dynamin: from fission to fusion

Nat Cell Biol

Fab1p is essential for PtdIns(3)P 5-kinase activity and the maintenance of vacuolar size and membrane homeostasis

J Cell Biol

PtdIns(3,5)P₂ and vacuole size regulation