Inositol pyrophosphates regulate endocytic trafficking Adolfo Saiardi*, Catherine Sciambi†, J. Michael McCaffery‡, Beverly Wendland†, and Solomon H. Snyder*§¶储 Departments of *Neuroscience, §Pharmacology and Molecular Sciences, and ¶Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205; and ‡Integrated Imaging Center, †Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218 Contributed by Solomon H. Snyder, August 30, 2002
The high energy potential and rapid turnover of the recently discovered inositol pyrophosphates, such as diphosphoinositolpentakisphosphate 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.
T
he recently identified inositol pyrophosphates diphosphoinositol-pent ak isphosphate (PP-IP 5 ; IP 7 ) and bisdiphosphoinositol-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兾peptone兾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 ipk1⌬ 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 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 14206 –14211 兩 PNAS 兩 October 29, 2002 兩 vol. 99 兩 no. 22
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. 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兾5 ml of ether兾1 ml of pyridine兾18 l of NH4OH), and incubating for 30 min at 57°C. The dry lipids were deacetylated in 0.5 ml of methylamine reagent (40% methylamine兾45% methanol兾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兾petroleum ether兾 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 l of prewarmed FM 4-64 dye (Molecular Probes) diluted 1:50 in YPD (FM 4-64 stock is 1 mg兾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兾ml stock diluted 1:50 in yeast
Abbreviations: IP6, inositol hexakisphosphate; IP6K, IP6 kinase; IP7, diphosphoinositolpentakisphosphate; IP8, bis-diphosphoinositol-tetrakisphosphate; IPMK, inositol phosphate multikinase. 储To
whom correspondence should be addressed. E-mail:
[email protected].
www.pnas.org兾cgi兾doi兾10.1073兾pnas.212527899
Strain BY4741 BY4742 YD03531 YD03674 YD03956 YD07135 YD05149 SEY6210 MBY3 BWY1205 BWY1206 BWY1207 BWY1212 BWY1213 BWY1214 BWY1224 BWY1225 BWY1226 BWY1227 BWY1228 BWY1516 BWY1340
Genotype
Source
MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0 MAT␣ his3⌬1 leu2⌬0 lys2⌬0 ura3⌬0 BY4741 arg82⬋kanMX4 BY4741 ipk1⬋kanMX4 BY4741 kcs1⬋kanMX40 BY4741 plc1⬋kanMX4 BY4741 vps34⬋kanMX4 MAT␣ leu2-3,112 ura3-52 his3-⌬200 trp1-⌬901 lys2-801 suc2-⌬9 SEY6210 vps4⬋TRP YD03531 ⫻ BY4742 MAT␣ arg82⬋kanMX4 from BWY1205 MATa arg82⬋kanMX4 from BWY1205 YD03956 ⫻ BY4742 MAT␣ kcs1⬋kanMX4 from BWY1212 MATa kcs1⬋kanMX4 from BWY1212 BWY1206 ⫹ pARG82.416 BWY1213 ⫹ pKCS1.415 YD03674 ⫻ BY4742 MAT␣ ipk1⬋kanMX4 from BWY1226 MATa ipk1⬋kanMX4 from BWY1226 BWY1214 ⫻ BWY1227 MATa ipk1⬋kanMX4 kcs1⬋kanMX4 from BWY1516
34 34 35 35 35 35 35 Wendland lab strain 36 This study This study This study This study This study This study This study This study This study This study This study This study This study
extract兾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 micros-
copy 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 Ste3⌬365-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兾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 (ip6k⌬), 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. 1). Normal intracellular membrane morphology and normal levels of IP7 and IP8 are restored by transforming the mutants with IP6K plasmids (Fig. Saiardi et al.
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 IP 3 (10, 17), and do not form either IP 6 or the pyrophosphates IP7 and IP8 (Fig. 1). They display morphological abnormalities similar to the ip6k⌬ mutant (Fig. 1). These perturbations are rescued by the introduction of an IPMK plasmid. The only biochemical defect common to both ipmk⌬ and ip6k⌬ 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 ipmk⌬ and ip6k⌬ yeast than WT. Quantitative analysis by f low cytometry indicates that this brighter signal ref lects augmented endocytosis, and not simply the superimposition of multiple vacuoles (see Table 2, which is published as supporting information on the PNAS web site). To explore further a role for inositol pyrophosphates in membrane trafficking, we examined mutants lacking inositol phosphate kinase 1 (IPK1; YDR315c; Fig. 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 ipk1⌬ 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 PNAS 兩 October 29, 2002 兩 vol. 99 兩 no. 22 兩 14207
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Table 1. Yeast strains used in this study
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 ipk1⌬ yeast, the Inset represents the HPLC analysis of the putative [PP]2-IP3, using recombinant diphosphoinositol polyphosphate phosphohydrolase protein (DDP1) (12). About 4,000 cpm of the purified 2(PP)-IP3 peak (filled circles) were incubated with 20 ng of recombinant DIPP proteins for 1 h at 37°C (open circles). We observed conversion of 2(PP)-IP3 to PP-IP4, reflecting loss of one of the pyrophosphates, and to IP5, reflecting loss of both pyrophosphate groups. The standards used to identify the inositol phosphates were: IP2, [3H]I(1,4)P2; IP3, [3H]I(1,4,5)P3; IP4, [3H]I(1,3,4,5)P4; IP5, [3H]I(1,3,4,5,6)P5; IP6, [3H]IP6; IP7, [3H]PP-IP5; IP8, [3H]2(PP)-IP4. Transformation of the ipmk⌬ and ip6k⌬ yeast with the plasmid carrying the deleted genes pIPMK and pIP6K, respectively, restored normal levels of inositol pyrophosphate and normal FM 4-64 internalization. (Right) The lipophilic dye FM 4-64 was used to label yeast membranes for 15 min; cells were then washed and chased for 60 min at 30°C. DIC, dicroic field microscopy. Images were acquired under identical conditions to compare fluorescent intensities.
of PP-IP4 and 2(PP)-IP3 in the ipk1⌬ mutant might ref lect 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 ipk1⌬ and ip6k⌬ (Fig. 1). This double mutant fails to form inositol pyrophosphates and 14208 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.212527899
accumulates membranous兾vesicular elements closely resembling the abnormalities in the ip6k⌬ and ipmk⌬ mutants. Thus, we conclude that the abnormal vesicular morphology is caused by the loss of inositol pyrophosphates, completely independent of IP6. Saiardi et al.
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. 2). PI(4,5)P2 levels are substantially decreased in the ip6k⌬ mutants and increased in the ipmk⌬ mutants. Because the ipmk⌬ and ip6k⌬ 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). We performed electron microscopy to characterize the nature of the membranous structures that accumulate in the inositol pyrophosphate mutants (Fig. 3). The ipmk⌬ and ip6k⌬ 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. 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 Saiardi et al.
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Fig. 2. Levels of PI(4,5)P2 do not correlate with aberrant membrane trafficking in ipmk⌬ and ip6k⌬ yeasts. HPLC analysis of inositol lipids extracted and deacetylated from early logarithmic growth of [3H]inositol-labeled yeast. Deacetylated PI(4,5)P2 was identified by co-elution with authentic standards. The deacetylated PI(3,5)P2 peak was identified by its migration relative to ADP, ATP, and PI(4,5)P2 standards. Data are presented as means ⫾ SE of three or four independent experiments.
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 ipmk⌬ and ip6k⌬ mutants, the morphology of ipk1⌬ mutants is similar to WT (Fig. 3). To clarify further the nature of the endosome-like vesicular structures accumulating in ipmk⌬ and ip6k⌬ 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 ipmk⌬ and ip6k⌬ mutants (Fig. 4A), confirming that they are indeed aberrant endosomal intermediates. 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 ip6k⌬ mutants by using the lipophilic dye FM 4-64 that labels membranes (Fig. 4B). 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 ip6k⌬ 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 liganddependent internalization and degradation of Ste3⌬365 in ip6k⌬ compared with WT yeast (Fig. 4C). The ip6k⌬ cells accumulate a smaller Ste3 fragment consistent with abnormal processing兾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 py rophosphates regulate clathrinassociated 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. 1, Table 2). Alternatively, interconversion between IP6 and IP7 could inf luence the conformation of clathrin-associated proteins in
Fig. 3. Abnormal membranous structures accumulate in ipmk⌬ and ip6k⌬ yeasts. Electron microscopy reveals abnormal membranous organelles that accumulate in the absence of inositol pyrophosphate biosynthetic enzymes. The morphology of ipk1⌬ yeast is indistinguishable from WT. By contrast, major alterations in ipmk⌬ and ip6k⌬ yeast are evident. *, stacked membranous cisternae; arrows, ER; #, aberrant organelles; n, nucleus; m, mitochondria; V, vacuole.
the same way that interconversion of GDP and GTP regulates G protein function. Regardless of detailed mechanisms, our findings establish that inositol pyrophosphates regulate endo14210 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.212527899
cytic events, presumably by inf luencing the interactions of clathrin adaptor proteins, clathrin, and plasma membrane phospholipids. Saiardi et al.
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.
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PNAS 兩 October 29, 2002 兩 vol. 99 兩 no. 22 兩 14211
CELL BIOLOGY
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 ipmk⌬ and ip6k⌬ mutants. (B) Time course of labeling with FM 4-64 in WT and ip6k⌬ yeast. After 15 min labeling on ice, cells were washed and incubated at 30°C for 5, 10, or 20 min. In the ip6k⌬ mutants after 20 min, the dye is still found in small bright membranous structures. (C) Turnover of the plasma membrane mating pheromone receptor Ste3 (15). The right panel represent a pulse– chase experiment where we observed a retardation of the processing of Ste3 (#) in ip6k⌬ yeast compared with WT with an accumulation of a smaller Ste3 fragment (⬍), reflecting altered processing of the protein and endosomal missorting. (Left) Quantification of three separate experiments. Transformation of the ip6k⌬ yeast with the plasmid carrying the deleted gene restored normal Ste3p processing. The asterisks denote significant retardation of Ste3p processing in ip6k⌬ yeast (P ⬍ 0.05, paired t test).
