In this study, we show that the PH domain-containing proteins Slm1 and Slm2 are functionally redundant and essential for cell growth and PtdIns(4,5)P2-dependent signaling to the actin cytoskeleton. We further demonstrate that Slm1 and Slm2 physically interact with Avo2 and Bit61 and are novel components of the TORC2 signaling complex. Slm1 (and Slm2) localize to the plasma membrane via interaction with PtdIns(4,5)P2 and TORC2. In addition, Tor2 positively and possibly directly regulates the phosphorylation of Slm1 and Slm2 suggesting that Slm function is modulated in response to signals that control Tor2. Collectively, our data suggest that Slm1 and Slm2 are novel PtdIns(4,5)P2 effectors that integrate inputs from the PtdIns(4,5)P2 and Tor2 signaling pathways to modulate polarized actin assembly and growth. We propose that the synergistic interaction with lipid and protein ligands ensures the proper localization and regulation of Slm1 and Slm2 and is important for the compartmentalization and differential effects of PtdIns(4,5)P2-dependent signaling events.
Our data suggest that Slm1 and Slm2 respond specifically to changes in PtdIns(4,5)P2
levels and thus constitute novel PtdIns(4,5)P2
effectors. The lipid binding activity of Slm1 and Slm2 resides in their C-terminal PH domains, and our analysis of Slm truncation and point mutants suggests that these domains serve as membrane anchors that direct the two proteins to sites enriched in their ligand PtdIns(4,5)P2
. This model is supported by a recent survey of yeast PH domains, which found that the isolated Slm1 and Slm2 PH domains localized to the plasma membrane in a manner that was dependent on PtdIns(4,5)P2
synthesis (Yu et al., 2004 )
. Although the PH domain of Slm1, like other PH domains, binds to many polyphosphoinositides with no apparent selectivity in vitro, our data further argue that in vivo, Slm proteins specifically bind PtdIns(4,5)P2
and that PtdIns(4,5)P2
binding is essential for Slm localization and function. First, targeting of Slm1 and Slm2 to the plasma membrane is dependent upon the integrity of their PH domains and requires lipid binding activity. Second, plasma membrane association of Slm1 and Slm2 is affected by changes in PtdIns(4,5)P2
levels such as in stt4ts
mutants, and in cells that express the phosphatase SigD, which hydrolyzes PtdIns(4,5)P2
. Third, manipulating cellular PtdIns(4,5)P2
levels by disruption of the PtdIns(4,5)P2
suppresses the lethality of slm
Δ null mutant cells. Fourth, slm1
Δ mutations are synthetically lethal with hypomorphic mutations in STT4
. Last, we demonstrate that the ability to bind PtdIns(4,5)P2
is essential for Slm function, because Slm mutants that fail to bind phosphoinositides in vitro, also lack the ability to complement the lethality of slm
Δ null mutant cells in vivo.
Although the PH domains of Slm1 and Slm2 are necessary and sufficient to direct plasma membrane targeting (Yu et al., 2004
; our unpublished observations), our data suggest that Slm proteins interact with a second, PtdIns(4,5)P2
-independent factor at the plasma membrane that directs Slm localization to specific plasma membrane subdomains. This notion is based on the finding that Slm proteins do not uniformly localize along the plasma membrane, as do isolated PH domains that bind PtdIns(4,5)P2
, but concentrate in discrete clusters. Our studies identify the TORC2 complex as a candidate PtdIns(4,5)P2
-independent localization component, although we cannot rule out contribution by additional factors. Slm1 and Slm2 physically interact with the TORC2 components Avo2, Bit61, and Ybr270c, and our findings from biochemical and immunofluorescence studies are consistent with a role of these proteins in stabilizing and possibly refining Slm localization at the plasma membrane. Such combinatorial control of protein targeting by both lipid and protein determinants may be a common mechanism to regulate the localization and function of PH domain-containing proteins. For example, the targeting of the mammalian four-phosphate adaptor proteins 1 and 2 (Fapp1 and Fapp2) to the Golgi is mediated by the simultaneous binding of their PH domains to PtdIns(4)P and the GTPase Arf1 (Godi et al., 2004
). PtdIns(4)P and a Golgi-localized factor also determine the targeting of the related PH domain-containing oxysterol binding proteins (Fang et al., 1996
; Levine and Munro, 2002
). Similarly, Cla4, Boi1, and Boi2 are targeted to sites of polarized growth by both lipid-dependent and -independent mechanisms (Bender et al., 1996
; Hallett et al., 2002
; Wild et al., 2004
). Such a synergistic control mechanism may explain why many PH domain-containing proteins in vitro show little specificity in phosphoinositide recognition and bind with only moderate affinity, but still target in a specific manner in vivo.
Like mutants in STT4, MSS4
, and TOR2, slm
deletion and temperature-sensitive mutants exhibit defects in actin cable assembly and actin patch polarization. Together with our biochemical data demonstrating a direct physical interaction between Slm proteins, PtdIns(4,5)P2
, and TORC2 components, these findings suggest that Slm proteins integrate inputs from Tor2 and PtdIns(4,5)P2
to modulate actin cytoskeleton polarization. Consistent with the observed cytoskeletal defects, we found that two proteins, Cdc42 and Rho1, which depend on actin cables for their polarized transport to and retention at sites of growth failed to efficiently concentrate in the growing bud upon loss of Slm activity. How do Slm proteins affect actin cytoskeleton dynamics? Because the precise molecular function of Slm1 and Slm2 is at present unknown, there are multiple ways of how a loss of Slm function might lead to the observed cytoskeletal defects. Two likely possibilities are that Slm proteins either directly affect actin cable assembly or that they play a role in vesicular trafficking to the plasma membrane. Rho1 and Cdc42 are key regulators of actin cytoskeletal dynamics that promote formin-dependent actin cable assembly (Tolliday et al., 2002
; Dong et al., 2003
) and their transport to the bud is mediated by vesicular transport along actin cables (Abe et al., 2003
; Wedlich-Soldner et al., 2003
; Pruyne et al., 2004
). Thus, a failure or decrease in their secretion may indirectly cause the observed cytoskeletal defects in slm
Δ mutants. Consistent with such a hypothesis, a block in secretion in several sec
mutants also causes actin cable and actin polarization defects (Mulholland et al., 1997
; Pruyne et al., 2004
). Regardless of the precise mechanism, the phenotypic suppression of slm
null mutants by overexpression of the cell integrity pathway component PKC1
suggests that Slm1 and Slm2 modulate Pkc1 activity or localization. Whether Slm1 and Slm2 signaling to Pkc1 involves the upstream activator Rho1 remains to be determined. Contrary to Rho1 and Cdc42, the localization of Pkc1 to the cell cortex is independent of vesicular trafficking (Andrews and Stark, 2000
), which may explain why PKC1
is a more effective suppressor of slm
null mutant phenotypes compared with RHO1
In yeast, signaling from Tor2p to the actin cytoskeleton bifurcates at the level of Pkc1p (Delley and Hall, 1999
). Interestingly, overexpression or mutational activation of components of the Pkc1-activated mitogen-activated protein (MAP) kinase cascade failed to suppress the phenotypes of slm
Δ null mutant cells, and no synthetic lethality was observed between slm1
Δ mutants and mutants in the MAP kinase module (our unpublished data). Thus, Slm signaling likely involves the MAP kinase independent Pkc1 signaling branch. This branch controls the cell cycle-dependent polarization of the actin cytoskeleton through yet to be defined effectors (Delley and Hall, 1999
). Alternatively, and equally consistent with our data, Slm1 and Slm2 may act in a pathway that has a function overlapping with Pkc1.
Future studies also need to address the precise roles of Slm proteins and the TORC2 components Avo2, Bit61, and Ybr270c in the Tor2 signaling pathway. Preliminary studies indicate that SLM
overexpression does not suppress the lethality of tor2ts
mutants and only marginally restores actin cable assembly in these mutants (our unpublished observations). However, suppression is not necessarily to be expected because most components of the TORC2 complex (except Avo2) are essential proteins (Loewith et al., 2002
), and mutants therein also exhibit actin defects (Loewith et al., 2002
). It is thus likely that Slm1 and Slm2 regulate a subset of TORC2 functions activated in response to specific environmental signals. Future identification of downstream effectors of Slm1 and Slm2 and characterization of the role of TORC2 in Slm1 and Slm2 regulation will likely provide insight into the exact molecular function of Slm proteins and reveal how they signal to the actin cytoskeleton.
Interestingly, the TORC2 complex and its modulation of PKC function may be conserved in mammalian cells. Two recent publications reported that the mammalian mTOR protein is part of a protein complex that contains the Avo3 homolog rictor (Jacinto et al., 2004
; Sarbassov dos et al., 2004
). This complex mediates rapamycin-insensitive mTOR signaling to the actin cytoskeleton (Jacinto et al., 2004
; Sarbassov dos et al., 2004
) through modulation of protein kinase Cα (Sarbassov dos et al., 2004
), suggesting that at least some aspects of TORC2 signaling are conserved between yeast and mammals.
In summary our studies identify two novel effectors of PtdIns(4,5)P2 that mediate an essential function required for growth and actin cytoskeletal organization. The yeast actin cytoskeleton is highly dynamic and quickly adapts to changes in various environmental conditions and in response to the cell cycle phase. Thus, actin dynamics is likely controlled and modulated by a network of signaling pathways that sense and integrate different stimuli. Slm1 and Slm2 contain lipid and protein binding modules and seem to function as part of such a signaling network to integrate signals from the Stt4/Mss4 and Tor2 pathways. In that way, PtdIns(4,5)P2 signaling is coordinated with and dependent on other signaling pathways such as the Tor2 pathway. This, in turn, may allow the differential regulation of PtdIns(4,5)P2-dependent signaling processes in response to various environmental and intracellular stimuli.
Note added in proof.
While this article was under review, S. Emr and colleagues (Audhya et al., 2004
) reported on the identification of Slm1 and Slm2 in a synthetic lethal screen with mss4ts. Their and our findings are in good agreement.