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The yeast phosphatidylinositol-4-OH kinase Pik1 regulates secretion at the Golgi

Nature Cell Biology volume 1, pages 523–525 (1999)Cite this article

Phosphoinositides participate in signal transduction, cytoskeletal organization and membrane traffic in eukaryotic cells. Phosphatidylinositol kinases, phosphatases and transfer proteins act in specific processes and stages of membrane transport1,2. A phosphatidylinositol-4-OH kinase (PtdIns-4-OH kinase) activity associated with small synaptic vesicles and chromaffin granules is required for stimulated secretion in mammalian cells3,4. In Saccharomyces cerevisiae, two PtdIns-4-OH kinases have been identified, namely Stt4, which acts in the pathway of the protein kinase Pkc1 (ref. 5), and Pik1, which participates in the mating-pheromone signal-transduction cascade6. As both are essential for growth6,7,8 they must have distinct functions. An involvement of Pik1 in secretion has so far not been described. We report here that Pik1 is a regulator of membrane traffic. The temperature-sensitive mutation pik1-101 results in defects in the actin cytoskeleton. However, blockages in membrane traffic at the Golgi apparatus and the endosome, as well as genetic evidence, show that Pik1 has an actin-independent role in membrane transport. We show that Pik1 localizes to the nucleus and cytoplasm, where it is concentrated at the trans-Golgi, in agreement with its function in the Golgi apparatus.

The small GTPase Sec4 regulates Golgi-to-plasma-membrane transport9. The GDP-dissociation inhibitor Gdi1 recycles GDP-bound Sec4 from the plasma membrane to secretory vesicles10. In a search for new regulators of secretion, we performed a genetic screen to isolate mutants that exhibit synthetic lethality upon overproduction of Gdi1. Mutations were introduced randomly into the genome with the mutagen ethylmethane sulphonate. One of these mutations showed an initial 44% block of secretion (at 37 °C) of the secretory-vesicle cargo invertase (after backcrossing, a 33 ± 2% block was observed; mean ± s.e.m.) and was located to the PIK1 gene by complementation cloning and linkage analysis. The pik1-101 mutation results in inositol auxotrophy. Sequence analysis showed that this mutant had a cytosine-to-thymine point mutation at position 3,137 of the PIK1 open reading frame, encoding a change of serine at position 1,045 to phenylalanine within the catalytic domain6.

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Figure 1: The pik1-101 mutant allele causes a defect in secretion at the Golgi apparatus, disturbs the actin cytoskeleton and results in low PtdIns-4-OH kinase activity.
Figure 2: Pik1 localizes to both the nucleus and the cytoplasm, where it concentrates at the trans-Golgi.

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Acknowledgements

This work was supported by grants from the NIH (to P.N.) and the Yale Diabetes Endocrinology Research Center (to C.W.-S.). We thank M. Solimena, P. De Camilli and C. M. Carr for critical reading of the manuscript; E. O’Neill and M. Travers for technical assistance; S. Wolin for the anti-Lhp1 antibody and discussions; M. Snyder for providing yeast strains containing 3× haemagglutinin-tagged Chs5; G.Waters for the haemagglutinin–OCH1 plasmid; and J. Valentijn for help with computer programs. Microscopy studies were partially carried out with support of the Yale Diabetes Endocrinology Research Center (DERC) Cell Biology Core.

Correspondence and requests for materials should be addressed to P.N.

Supplementary information is available on Nature Cell Biology’s World-Wide Web site (http://cellbio.nature.com) or as paper copy from the London editorial office of Nature Cell Biology.

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  1. Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, 06510, Connecticut, USA

    Christiane Walch-Solimena & Peter Novick

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  1. Christiane Walch-Solimena
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Correspondence to Peter Novick.

Supplementary information

Figure S1

The pik1-101 mutant allele blocks endocytosis at the endosome. (PDF 714 kb)

Figure S2

Characterization of the anti-Pik1p antibody. (PDF 100 kb)

Figure S3

The nuclear proteins Pik1p and Lhp1p translocate rapidly from the nucleus to the cytoplasm upon a block in secretion. (PDF 1648 kb)

Table S1

Viability of pik1-101 double mutants. (PDF 44 kb)

Table S2

Relevant yeast strains. (PDF 3 kb)

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Walch-Solimena, C., Novick, P. The yeast phosphatidylinositol-4-OH kinase Pik1 regulates secretion at the Golgi. Nat Cell Biol 1, 523–525 (1999). https://doi.org/10.1038/70319

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