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Protein Ser/Thr phosphatase-1 (PP1) catalyzes the majority of eukaryotic protein dephosphorylation reactions in a highly regulated and selective manner. Recent studies have identified an unusually diversified PP1 interactome with the properties of a regulatory toolkit. PP1-interacting proteins (PIPs) function as targeting subunits, substrates and/or inhibitors. As targeting subunits, PIPs contribute to substrate selection by bringing PP1 into the vicinity of specific substrates and by modulating substrate specificity via additional substrate docking sites or blocking substrate-binding channels. Many of the nearly 200 established mammalian PIPs are predicted to be intrinsically disordered, a property that facilitates their binding to a large surface area of PP1 via multiple docking motifs. These novel insights offer perspectives for the therapeutic targeting of PP1 by interfering with the binding of PIPs or substrates.

SUMMARY Macroautophagy is a bulk clearance mechanism in which the double-membraned phagophore grows and engulfs cytosolic material. In yeast, the phagophore nucleates from a cluster of 20-30 nm diameter Atg9-containing vesicles located at a multiprotein assembly known as the preautophagosomal structure (PAS). The crystal structure of a 2:2:2 complex of the earliest-acting PAS proteins, Atg17, Atg29, and Atg31, was solved at 3.05 Å resolution. Atg17 is crescent-shaped with a 10 nm radius of curvature. Dimerization of the Atg17-Atg31-Atg29 complex is critical for both PAS formation and autophagy and each dimer contains two separate and complete crescents. Upon induction of autophagy, Atg17-Atg31-Atg29 assembles with Atg1 and Atg13, which in turn initiates the formation of the phagophore. The C-terminal EAT domain of Atg1 was shown to sense membrane curvature, dimerize, and tether lipid vesicles. These data suggest a structural mechanism for the organization of Atg9 vesicles into the early phagophore.

The serine/threonine Protein Phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets. PP1 associates with ≥200 regulatory proteins to form highly specific holoenzymes. These regulatory proteins target PP1 to its point of action within the cell and prime its enzymatic specificity for particular substrates. However, how they direct PP1’s specificity is not understood. Here we show that spinophilin, a neuronal PP1 regulator, is entirely unstructured in its unbound form and binds PP1, through a folding-upon-binding mechanism, in an elongated fashion, blocking one of PP1’s three putative substrate binding sites, without altering its active site. This mode of binding is sufficient for spinophilin to restrict PP1’s activity toward a model substrate in vitro, without affecting its ability to dephosphorylate its neuronal substrate GluR1. Thus, our work provides the molecular basis for the ability of spinophilin to dictate PP1 substrate specificity.

SUMMARY Macroautophagy is essential to cell survival during starvation and proceeds by the growth of a double-membraned phagophore, which engulfs cytosol and other substrates. The synthesis and recognition of the lipid phosphatidylinositol 3-phosphate (PI(3)P) is essential for autophagy. The key autophagic PI(3)P sensors, which are conserved from yeast to humans, belong to the PROPPIN family. Here we report the crystal structure of the yeast PROPPIN Hsv2. The structure consists of a seven-bladed β-propeller, and unexpectedly, contains two pseudo-equivalent PI(3)P binding sites on blades 5 and 6. These two sites both contribute to membrane binding in vitro and are collectively required for full autophagic function in yeast. These sites function in concert with membrane binding by a hydrophobic loop in blade 6, explaining the specificity of the PROPPINs for membrane-bound PI(3)P. These observations thus provide a structural and mechanistic framework for one of the conserved central molecular recognition events in autophagy.

Emerging evidence suggests that myocyte enhancer factor 2 (MEF2) transcription factors act as effectors of neurogenesis in the brain, with MEF2C the predominant isoform in developing cerebrocortex. Here, we show that conditional knockout of Mef2c in nestin-expressing neural stem/progenitor cells (NSCs) impaired neuronal differentiation in vivo, resulting in aberrant compaction and smaller somal size. NSC proliferation and survival were not affected. Conditional null mice surviving to adulthood manifested more immature electrophysiological network properties and severe behavioral deficits reminiscent of Rett syndrome, an autism-related disorder. Our data support a crucial role for MEF2C in programming early neuronal differentiation and proper distribution within the layers of the neocortex.neurogenesis ͉ synaptogenesis ͉ autism ͉ Rett syndrome K nockdown of the transcription factor MEF2C in mature cerebrocortical neurons results in increased synaptic number and activity (1). To facilitate analysis of MEF2C function in early neuronal development, we engineered a conditional knockout in NSCs by crossing floxed Mef2c mice with Nestin-Cre mice. In contrast to the findings in more mature neurons, we found a striking alteration in the distribution of new neurons in the neocortex and the opposite effect on synaptic activity, i.e., decreased neurotransmission persisting into adulthood.MEF2C belongs to the myocyte enhancer factor 2 (MEF2) subfamily of the MADS (MCM1-agamous-deficiens-serum response factor) gene family (2, 3). We cloned MEF2C from developing mouse brain, and Eric Olson and colleagues then discovered it in the heart (2, 4, 5). In cerebrocortex, MEF2 transcriptional activity is restricted to differentiated cortical neurons in a specific laminar pattern, and its distribution increases along the rostrocaudal axis (2, 4, 6). These features led to speculation on the potential role of MEF2C in the architechtonics of the cerebral cortex (2). Previous studies demonstrated an important role for MEF2C in heart development (7). In the CNS, MEF2C is involved in neuronal apoptosis (8) and synapse formation (1, 9) in vitro or in brain slices. Most recently, our laboratory discovered that a constitutively active form of MEF2C induces embryonic stem cells to commit to a neuronal fate in a virtually exclusive fashion (10). However, studies on the effect of endogenous MEF2C on CNS neurons in vivo were impeded by the embryonic lethality of conventional Mef2c-null mice because of cardiovascular defects at embryonic day (E) 9.5, before brain development (7). Here, we report that conditionally knocking out the Mef2c gene in neural progenitors causes abnormal aggregation and compaction of neurons migrating into the lower layers of the neocortex during development. Knockout mice surviving to adulthood manifest smaller, apparently less mature neurons and smaller whole brain size, with resultant aberrant electrophysiology and behavior.

Complete folding is not a prerequisite for protein function, as disordered and partially folded states of proteins frequently perform essential biological functions. In order to understand their functions at the molecular level, we utilized diverse experimental measurements to calculate ensemble models of three non-homologous, intrinsically disordered proteins: I-2, spinophilin and DARPP-32, which bind to and regulate protein phosphatase 1 (PP1). The models demonstrate that these proteins have dissimilar propensities for secondary and tertiary structure in their unbound forms. Direct comparison of these ensemble models with recently determined PP1 complex structures suggests a significant role for transient, pre-formed structure in the interactions of these proteins with PP1. Finally, we generated an ensemble model of partially disordered I-2 bound to PP1 that provides insight into the relationship between flexibility and biological function in this dynamic complex.