Phosphoinositides (PI) control numerous cellular processes including cell signaling, growth, vesicular trafficking, and actin cytoskeletal arrangements (8
). In particular, phosphatidylinositol(4,5)-bisphosphate [PI(4,5)P2
] provides important regulatory roles at the cell surface, as a precursor of the signaling molecules DAG and IP3
, and through direct interactions with downstream effector proteins (31
). During endocytic internalization, PI(4,5)P2
recruits or activates several proteins, including the clathrin adaptor complex AP-2, AP180, epsin, and the GTPase dynamin, leading to the formation and fission of clathrin-coated vesicles (13
also recruits or controls several actin-regulatory proteins (54
). Actin polymerization may provide a propulsive force to facilitate membrane invagination and fission events or to transport newly formed endocytic vesicles away from the plasma membrane. Thus, PI(4,5)P2
synthesis plays several important roles in the formation and fission of clathrin-coated vesicles during endocytosis.
Likewise, previous studies implicate dephosphorylation of PI(4,5)P2
by the polyphosphoinositide phosphatase (PPIPase) synaptojanin as an important step in endocytic internalization (7
). Synaptojanins contain two distinct PI phosphatase domains and a C-terminal proline-rich domain (PRD). The N-terminal Sac1 domain (PPIPase domain) acts on several positions of the inositol ring, while its central phosphatase domain specifically acts at the 5′ position (5-Pase domain) (20
). The PRD region functions as a targeting domain via interactions with various SH3 domain-containing proteins, such as endophilin, amphiphysin, and CIN85 (5
). Coated vesicles accumulate in cells bearing genetic disruptions of synaptojanin isoforms in the mouse, Drosophila melanogaster
, and Caenorhabditis elegans
, suggesting that turnover of PI(4,5)P2
mediates release of coat proteins from membranes of newly formed vesicles (7
). Since synaptojanins regulate PI(4,5)P2
-mediated actin polymerization, they may also control earlier vesicle formation and/or fission events, along with their role in postfission vesicle uncoating.
Three synaptojanin-like proteins are present in Saccharomyces cerevisiae
: Sjl1, Sjl2, and Sjl3 (also named Inp51, Inp52, and Inp53) (46
). Like mammalian synaptojanin, Sjl2 and Sjl3 exhibit Sac1 PPIPase activities, and all three possess 5-Pase activity (Fig. ) (20
). Factors that target the yeast synaptojanin-like proteins via their PRDs have yet to be demonstrated in vivo. Interestingly, the C-terminal domains of Sjl1, Sjl2, and Sjl3 do not share extensive identity (Fig. ). Thus, while synaptojanins display similar activities in vitro, each may provide specialized functions in vivo and may be uniquely recruited or activated at distinct membrane sites.
FIG. 1. Yeast synaptojanin-like proteins possess unique C-terminal domains. The Sac1 domains in Sjl1p, Sjl2p, and Sjl3p are shown by open ovals. The asterisks in the Sac1-like domain of Sjl1 indicate that it does not possess PPIPase activity. Dark gray bars, (more ...)
Previous studies have indicated that Sjl2 provides essential overlapping functions at both the plasma membrane and intracellular endosomal/late Golgi compartments (17
). Moreover, we have shown that yeast cells expressing only a temperature-conditional SJL2
allele inappropriately accumulate PI(4,5)P2
in intracellular compartments (48
). These findings have thus led to the question of whether Sjl2 acts primarily at the plasma membrane or at endosomes in the control of PI(4,5)P2
steady-state distribution. To address this question, we have examined the localization of Sjl2 in vivo. Sjl2 was recruited to cortical actin patches in a manner dependent on filamentous actin and the PRD of Sjl2. We further addressed how Sjl2 is recruited to its site of action at the plasma membrane and found that the SH3-containing protein Abp1 was essential for this process. Finally, we found that overexpression of the ANTH domain-containing protein Sla2 can partially suppress the endocytic defects in sjl1
Δ sjl2ts sjl3
Δ mutant cells, suggesting that Sla2 becomes a limiting factor during endocytic internalization in cells with impaired PI 5-Pase activity.