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Proc Natl Acad Sci U S A. Oct 29, 2002; 99(22): 14206–14211.
Published online Oct 21, 2002. doi:  10.1073/pnas.212527899
PMCID: PMC137862
Cell Biology

Inositol pyrophosphates regulate endocytic trafficking

Abstract

The high energy potential and rapid turnover of the recently discovered inositol pyrophosphates, such as diphosphoinositol-pentakisphosphate and bis-diphosphoinositol-tetrakisphosphate, suggest a dynamic cellular role, but no specific functions have yet been established. Using several yeast mutants with defects in inositol phosphate metabolism, we identify dramatic membrane defects selectively associated with deficient formation of inositol pyrophosphates. We show that this phenotype reflects specific abnormalities in endocytic pathways and not other components of membrane trafficking. Thus, inositol pyrophosphates are major regulators of endocytosis.

The recently identified inositol pyrophosphates diphosphoinositol-pentakisphosphate (PP-IP5; IP7) and bis-diphosphoinositol-tetrakisphosphate [2(PP)-IP4; IP8] (1, 2) contain energetic pyrophosphate bonds and turn over rapidly (3), suggesting dynamic roles with potential molecular switch activity. However, cellular functions for these substances have been elusive. Actions related to intracellular vesicles are suggested by the vacuolar abnormalities in yeast lacking the biosynthetic enzyme inositol hexakisphosphate kinase (IP6K, also known as KCS1; YDR017c; ref. 4), binding of inositol hexakisphosphate (IP6) to clathrin-associated proteins (5–8), and the associations of mammalian IP6K with a recently identified protein GRAB (guanine nucleotide exchange for Rab3A), a physiological guanine nucleotide exchange factor for Rab3A, a protein that mediates synaptic vesicle release (9). Using mutants of the inositol pyrophosphate biosynthetic pathway in yeast, we now establish a selective role for inositol pyrophosphates in endocytic trafficking.

Media and Materials

Yeast were grown in standard yeast extract/peptone/dextrose (YPD) or in synthetic medium supplemented with the appropriate amino acid mixture Synthetic Complete Supplement mixture (SC) purchased from either Qbiogene (Carlsbad, CA) or Fisher Scientific. [3H]I(1)P1, [3H]I(1,4)P2, [3H]I(1,4,5)P3, [3H]I(1,3,4,5)P4, [3H]IP6, and [3H]PI(4,5)P2 were from Perkin–Elmer NEN. [3H]I(1,3,4,5,6)P5 was prepared by phosphorylating [3H]Ins(1,3,4,5)P4, using yIPMK (IMPK, inositol phosphate multikinase) (10). [3H]IP7 was prepared by phosphorylation of [3H]InsP6, using mouse IP6K1 (4). [3H]PP-IP4 was purified from [3H]inositol-labeled ipk1Δ yeast. All of the radiolabeled inositol phosphates synthesized were purified by HPLC as described (see below) and desalted (2).

Plasmids and Strain Construction.

The strains used in this study, some of which were generated by standard mating and tetrad dissection, are listed in Table Table1.1. Standard recombinant DNA techniques were performed as described (11). PCR of genomic DNA from the yeast WT strain BY4741 (Research Genetics, Huntsville, AL) was used to clone the genes used in this study. Yeast IP6K gene (YDR017c) was amplified using the oligos 5′-GCTGCGGCCGCTTTAACCTTAAACCAAACAT-3′ and 5′-GCTGCGGCCGCATGTACATATATCCTCACA-3′ to obtain an amplified sequence from 382 nt upstream to 351 nt downstream of the protein coding region and cloned in the NotI site of pRS415 plasmid. Yeast IPMK gene (YDR173c) was amplified using the oligos 5′-GCTGCGGCCGCGGTGTGACAGGCTTGTTGTG-3′ and 5′-GCTGCGGCCGCATTTCTTGCAAACATAAGTA-3′ to obtain an amplified sequence from 440 nt upstream to 183 nt downstream of the protein coding region and cloned in the NotI site of pRS415 plasmid.

Table 1.
Yeast strains used in this study

The yeast diphosphoinositol polyphosphate phosphohydrolase (DDP1; ORF, YOR163w) was PCR amplified using the oligos 5′-CGaCTGCAGACATGGGCAAAACCGCGGATAATC-3′ and 5′-GCAGAATTCCTATTTGTCGTCTTTAATGAT-3′ and subcloned in the PstI and EcoRI sites of the pTrcHisB expression vector (Invitrogen). The 5′ end (amino acids 1–295) of S.c. YAP1801 (YHR161C) was PCR amplified using the oligos 5′-GCAGTCGACGATGACAACATATTTCAAG-3′ and 5′-GCTGCGGCCGCTTACATATCAATTAAATTGAG-3′ and subcloned in the SalI–NotI sites of the prokaryotic expression vector pGEX 4T-2 (Amersham Pharmacia Biotech).

High-Performance Liquid Chromatography Analysis of Inositol Phosphates.

Analysis of soluble inositol phosphates were performed as described (4, 10). Detailed methods can be found in Supporting Materials and Methods, which is published as supporting information on the PNAS web site, www.pnas.org. Inositol phosphates were identified by their co-elution with specific standards. Analysis of the putative [PP]2-IP3 was performed using the yeast homolog of human DDPI as described (12, 13).

Inositol lipids were extracted by resuspending the cell pellet in 0.5 ml of water, adding 0.7 ml of extraction buffer (15 ml of ethanol/5 ml of ether/1 ml of pyridine/18 μl of NH4OH), and incubating for 30 min at 57°C. The dry lipids were deacetylated in 0.5 ml of methylamine reagent (40% methylamine/45% methanol/11% l-butanolo) for 60 min at 53°C. The samples were dried, resuspended in 0.5 ml of water, and extracted three times with 0.5 ml of l-butanolo/petroleum ether/ethylformate (20:4:1). SpeedVac (Savant) dried samples were resuspended in water, and ≈2 × 106 cpm were HPLC resolved (14) by using a 4.6 × 125 mm PartiSphere SAX column (Whatman). The column was eluted with a gradient generated by mixing water and buffer B [1.3 M (NH4)2 HPO4, pH 3.8] as follows: 0–5 min, 0% B; 5–65 min, 0–30% B; 65–80 min, 30–100% B; 80–95 min, 100% B. Spectrophotometric monitoring of AMP, ADP, and ATP elution time and genuine deacetylated [3H]PI(4,5)P2 (Perkin–Elmer NEN) were used as standards.

FM 4-64 Analysis.

Cells were grown to mid-log phase in selective medium or YPD at 30°C. Cells (1 ml) were pelleted at 300 × g for 1 min and resuspended in 50 μl of prewarmed FM 4-64 dye (Molecular Probes) diluted 1:50 in YPD (FM 4-64 stock is 1 mg/ml in DMSO). After 15 min of labeling at 30°C, the cells were washed, chased for 60 min at 30°C, and observed. For the FM 4-64 time-course experiment, mid-log phase cells were labeled with FM 4-64 [1 mg/ml stock diluted 1:50 in yeast extract/peptone (YP)] on ice for 15 min. For the 0 min time point, cells were washed with ice-cold YP and held on ice. Other samples were washed with ice-cold YP, incubated at 30°C for 5, 10, or 20 min, washed with ice-cold YP, and held on ice. All images were acquired at identical exposures by using a DeltaVision deconvolving microscope (Applied Precision, Issaguah, WA) with a cooled charge-coupled device camera and processed identically by using PHOTOSHOP 7.0 (Adobe Systems, Mountain View, CA).

Electron Microscopy.

Conventional and immunoelectron microscopy were performed as described (15). Detailed methods can be found in Supporting Materials and Methods.

Ste3 Stabilization Assay.

Cells expressing a galactose-regulated, ligand-dependent Ste3Δ365-myc (15) were grown at 26°C to mid-log phase in minimal medium lacking uracil plus 0.1% yeast extract and 4% raffinose. Expression was induced by adding 3% galactose for 90 min. Each strain (2 × 5 OD600 equivalents) was harvested and resuspended in minimal medium lacking uracil plus 3% glucose for 30 min to stop new synthesis of Ste3-myc and to allow its accumulation at the plasma membrane. After collecting the 0-min time point, cells received supernatant from cells overexpressing either a factor or alpha factor, and aliquots were collected at 30-, 60-, 90-, and 120-min intervals. Cell extracts were separated on a SDS/10% PAGE, immunoblotted with the myc 9E10 monoclonal antibody and goat anti-mouse peroxidase, developed using Super Signal (Pierce), and analyzed by quantitative chemiluminescence with a ChemiImager (Alpha Innotech, San Leandro, CA).

Results and Discussion

In budding yeast lacking IP6K (ip6kΔ), the pyrophosphates IP7 and IP8 are not formed, and the yeast display pronounced accumulation of membranous vesicular structures that derive from the plasma membrane, evidenced by labeling with the lipophilic dye FM 4-64 (Fig. (Fig.1).1). Normal intracellular membrane morphology and normal levels of IP7 and IP8 are restored by transforming the mutants with IP6K plasmids (Fig. (Fig.1).1). The fact that mutant yeast depleted of IP7 have normal IP6 levels, yet still display major alterations in endocytic organelles, indicates a selective role for inositol pyrophosphates, rather than IP6, in vesicular trafficking. Inositol phosphate multikinase (IPMK; YDR173c; also known as ARG82 or ARGRIII) displays a broad substrate specificity, phosphorylating I(1,4,5)P3 (IP3) and I(1,3,4,5)P4 (IP4). IPMK can also form inositol pyrophosphates by converting I(1,3,4,5,6)P5 (IP5) to diphosphoinositol-tetrakisphosphate (PP-IP4) (10, 16–18). Yeast lacking IPMK accumulate inositol bisphosphate (IP2) and IP3 (10, 17), and do not form either IP6 or the pyrophosphates IP7 and IP8 (Fig. (Fig.1).1). They display morphological abnormalities similar to the ip6kΔ mutant (Fig. (Fig.1).1). These perturbations are rescued by the introduction of an IPMK plasmid. The only biochemical defect common to both ipmkΔ and ip6kΔ mutants is the loss of inositol pyrophosphates, suggesting that they are responsible for the morphological abnormalities. Intracellular compartments labeled by FM 4-64 are substantially brighter in ipmkΔ and ip6kΔ yeast than WT. Quantitative analysis by flow cytometry indicates that this brighter signal reflects augmented endocytosis, and not simply the superimposition of multiple vacuoles (see Table 2, which is published as supporting information on the PNAS web site).

Fig 1.
Abnormal accumulation of membranous structures in the absence of inositol pyrophosphates. (Left) HPLC analyses of inositol phosphates in [3H]inositol-labeled yeast by using a PartiSphere SAX column. In ipk1Δ yeast, the Inset represents ...

To explore further a role for inositol pyrophosphates in membrane trafficking, we examined mutants lacking inositol phosphate kinase 1 (IPK1; YDR315c; Fig. Fig.1).1). IPK1 carries out a single step in inositol phosphate metabolism, acting after IPMK and before IP6K to convert IP5 to IP6 (19, 20). Deletion of IPK1 leads to the absence of IP6, accumulation of IP5, and the formation of large amounts of two inositol pyrophosphates, PP-IP4 and bis-diphosphoinositol-triphosphate [2(PP)-IP3]. The morphology of ipk1Δ mutants is identical to WT cells despite major abnormalities in inositol phosphate metabolism, suggesting that the pyrophosphates PP-IP4 and 2(PP)IP3 mediate functions normally carried out by IP7 and IP8. Consistent with this, we found that the epsin N-terminal homology (ENTH) domain of yAP180A binds PP-IP4 with an affinity similar to IP6 and IP7 (see Fig. 5, which is published as supporting information on the PNAS web site). The high levels of PP-IP4 and 2(PP)-IP3 in the ipk1Δ mutant might reflect activity of IP6K, which can act on IP5 as well as IP6 (4). To prevent the formation of PP-IP4 and 2(PP)-IP3, we made a double mutant lacking both ipk1Δ and ip6kΔ (Fig. (Fig.1).1). This double mutant fails to form inositol pyrophosphates and accumulates membranous/vesicular elements closely resembling the abnormalities in the ip6kΔ and ipmkΔ mutants. Thus, we conclude that the abnormal vesicular morphology is caused by the loss of inositol pyrophosphates, completely independent of IP6.

Because PI(4,5)P2 regulates membrane trafficking (21–24) and is the precursor of the water-soluble inositol phosphates (25), we also monitored phosphoinositide levels in inositol pyrophosphate mutant strains (Fig. (Fig.2).2). PI(4,5)P2 levels are substantially decreased in the ip6kΔ mutants and increased in the ipmkΔ mutants. Because the ipmkΔ and ip6kΔ mutants display the same morphologic abnormalities but opposite alterations in PI(4,5)P2, it is unlikely that PI(4,5)P2 is responsible for the aberrant morphology of the mutants. Because our HPLC system does not resolve PI(3)P and PI(4)P, we monitored their sum, which does not differ significantly between WT and mutants (data not shown).

Fig 2.
Levels of PI(4,5)P2 do not correlate with aberrant membrane trafficking in ipmkΔ and ip6kΔ yeasts. HPLC analysis of inositol lipids extracted and deacetylated from early logarithmic growth of [3H]inositol-labeled yeast. ...

We performed electron microscopy to characterize the nature of the membranous structures that accumulate in the inositol pyrophosphate mutants (Fig. (Fig.3).3). The ipmkΔ and ip6kΔ mutants display stacked cisternae reminiscent of the “Class E” multilamellar endosomal compartment (26) that presumably correspond to the dye-labeled structures observed by light microscopy in these mutants (Fig. (Fig.1).1). Additionally, the mutants accumulate many cytoplasmic dot-like membranous particles. Whereas the endoplasmic reticulum (ER) in WT yeast is generally nondescript and situated parallel to the plasma membrane, ER membranes in the mutants appear hypertrophied and often perpendicular. Although this suggests that inositol pyrophosphates may also play a role in regulating the ER, secretion assays indicate that ER function is unaffected in our mutant strains (see Fig. 6D, which is published as supporting information on the PNAS web site). In contrast to the substantial abnormalities observed in the ipmkΔ and ip6kΔ mutants, the morphology of ipk1Δ mutants is similar to WT (Fig. (Fig.3).3).

Fig 3.
Abnormal membranous structures accumulate in ipmkΔ and ip6kΔ yeasts. Electron microscopy reveals abnormal membranous organelles that accumulate in the absence of inositol pyrophosphate biosynthetic enzymes. The morphology of ipk1Δ ...

To clarify further the nature of the endosome-like vesicular structures accumulating in ipmkΔ and ip6kΔ mutants, we carried out immunoelectron microscopy by using immunogold localization with antibodies to Pep12, an endosomal t-SNARE, and Vph1, a vacuolar H+-ATPase subunit that is also associated with endosomal membranes. Both antibodies specifically recognize membranes that accumulate in ipmkΔ and ip6kΔ mutants (Fig. (Fig.44A), confirming that they are indeed aberrant endosomal intermediates.

Fig 4.
Aberrant endosomal compartments and processing in cells lacking inositol pyrophosphates. (A) Immunoelectron microscopy with antibodies against the endosomal markers Pep12 and Vph1. Both antibodies specifically recognize membranes accumulated in ipmkΔ ...

The organelle abnormalities associated with diminished inositol pyrophosphate formation indicate a selective abnormality in the endocytic pathway. During endocytosis, the plasma membrane invaginates to form an endocytic vesicle that is transferred to an endosomal compartment that subsequently fuses with vacuoles. We monitored this process in WT and ip6kΔ mutants by using the lipophilic dye FM 4-64 that labels membranes (Fig. (Fig.44B). In WT yeast, the dye initially is associated with the plasma membrane, then moves to the interior of the yeast, and by 20 min labels ring-like vacuoles, with the dye inside on the limiting membrane. By contrast, labeling in the ip6kΔ mutants is found predominantly in a large number of dense aggregates juxtaposed to the vacuoles, even after 20 min, suggesting slowed kinetics of the dye transport in the mutant. Instead of fusing with vacuoles, the endosomes appear to form the large multilamellar intermediates seen by electron microscopy.

To assess endocytosis by an independent method, we monitored the turnover of the plasma membrane mating pheromone receptor Ste3 (15) in pulse–chase experiments. Consistent with a reduced rate of transit from endosomes to the vacuole, we observed a substantial retardation of the ligand-dependent internalization and degradation of Ste3Δ365 in ip6kΔ compared with WT yeast (Fig. (Fig.44C). The ip6kΔ cells accumulate a smaller Ste3 fragment consistent with abnormal processing/endosomal missorting. In contrast, markers of biosynthetic membrane trafficking pathways (27, 28), including transport and processing of the vacuolar hydrolases carboxypeptidase Y (CPY), alkaline phosphatase (ALP), and aminopeptidase I (API), are similar in WT and inositol pyrophosphate-depleted yeast (see Fig. 6).

Our findings establish that inositol pyrophosphates are essential for proper endocytic trafficking. Disordered endosomal processing and related morphological abnormalities occur selectively in mutants lacking inositol pyrophosphates.

How might inositol pyrophosphates influence endocytic trafficking? PI(4,5)P2 is thought to regulate vesicle processing by binding to clathrin-associated proteins such as AP2 and AP180 (29–31), which also bind IP6 and IP7 with high affinity and specificity (6–8). We observe high-affinity binding of [3H]IP7 and [3H]PP-IP4 to yeast AP180 (YAP1801; YHR161c). IC-50 values for IP6 in competing for ligand binding are 0.58, 1.1, and 1.7 nM for [3H]IP7, [3H]PP-IP4, and [3H]IP6, respectively (see Fig. 5).

How do inositol pyrophosphates regulate clathrin-associated proteins? IP7 and IP8 might compete with PI(4,5)P2 for binding to the epsin N-terminal homology (ENTH) domain-containing proteins of the endocytic machinery (32, 33); in the absence of IP7 and IP8, ENTH domains might bind PI(4,5)P2 more efficiently, thus promoting the augmented endocytosis we observed (Fig. (Fig.1,1, Table 2). Alternatively, interconversion between IP6 and IP7 could influence the conformation of clathrin-associated proteins in the same way that interconversion of GDP and GTP regulates G protein function. Regardless of detailed mechanisms, our findings establish that inositol pyrophosphates regulate endocytic events, presumably by influencing the interactions of clathrin adaptor proteins, clathrin, and plasma membrane phospholipids.

Supplementary Material

Supporting Information:

Acknowledgments

We thank A. Riccio, R. Claudio Aguilar, and J. Baggett for reading the manuscript and for helpful comments; H. C. Ha, E. Nagata, H. R. Luo, A. Resnick, and K. J. Hurt for suggestions and discussions; A. Snowman for technical assistance; T. Wei for help with the flow cytometry; and D. Klionsky and S. Emr for antisera. This work was supported by U.S. Public Health Service Grant MH18501 and Research Scientist Award DA00074 (to S.H.S.), National Institutes of Health Grant GM60979 (to B.W.), and National Science Foundation Grant DBI 0099705 (to J.M.M. and B.W.). B.W. is also supported by a Burroughs Wellcome Fund New Investigator in the Pharmacological Sciences and is a March of Dimes Basil O'Connor Scholar.

Abbreviations

  • IP6, inositol hexakisphosphate
  • IP6K, IP6 kinase
  • IP7, diphosphoinositol-pentakisphosphate
  • IP8, bis-diphosphoinositol-tetrakisphosphate
  • IPMK, inositol phosphate multikinase

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