The Phosphoinositide Phosphatase Sjl2 Is Recruited to Cortical Actin Patches in the Control of Vesicle Formation and Fission during Endocytosis

Abstract

The Saccharomyces cerevisiae synaptojanin-like proteins (Sjl1, Sjl2, and Sjl3) are phosphoinositide (PI) phosphatases that regulate PI metabolism in the control of actin organization and membrane trafficking. However, the primary sites of action for each of the yeast synaptojanin-like proteins remain unclear. In this study, we show that Sjl2 is localized to cortical actin patches, sites of endocytosis. Cortical recruitment of Sjl2 requires the actin patch component Abp1. Consistent with this, the SH3 domain-containing protein Abp1 physically associates with Sjl2 through its proline-rich domain. Furthermore, abp1Δ mutations confer defects resembling loss of SJL2; sjl1Δ abp1Δ double-mutant cells exhibit invaginated plasma membranes and impaired endocytosis, findings similar to those for sjl1Δ sjl2Δ mutant cells. Thus, Abp1 acts as an adaptor protein in the localization or concentration of Sjl2 during late stages of endocytic vesicle formation. Overexpression of the Hip1-related protein Sla2 delayed the formation of extended plasma membrane invaginations in sjl2^ts cells, indicating that Sla2 may become limiting or misregulated in cells with impaired PI phosphatase activity. Consistent with this, the cortical actin patch protein Sla2 is mislocalized in sjl1Δ sjl2Δ mutant cells. Together, our studies suggest that PI metabolism by the synaptojanin-like proteins coordinately directs actin dynamics and membrane invagination, in part by regulation of Sla2.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

We thank David Drubin, David Botstein, and Greg Odorizzi for providing strains and plasmids. We are grateful to Perla Arcaira for helpful technical assistance. We thank members of the Emr lab, especially William Parrish, for useful discussions. We thank Ingrid Niesman for assistance with the electron microscopic analysis (Immunoelectron Microscopy Core, headed by M. Farquhar).

C.J.S. was supported as a fellow of the American Cancer Society by the Holland Peck Charitable Fund and as an associate of the Howard Hughes Medical Institute. A.A. is currently supported by a Helen Hay Whitney postdoctoral fellowship. S.D.E. is an established investigator of the Howard Hughes Medical Institute.