Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here, we describe a single molecule pull-down (SiMPull) assay that combines the principles of conventional pull-down assay with single molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signaling proteins found in cytosol, membrane, and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled down proteins are functional and are used, without further processing, for single molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analyzing protein assemblies in biological pathways.

The endoplasmic reticulum (ER) and mitochondria form contacts that support communication between these two organelles, including synthesis and transfer of lipids, and the exchange of calcium, which regulates ER chaperones, mitochondrial ATP production, and apoptosis. Despite the fundamental roles for ER-mitochondria contacts, little is known about the molecules that regulate them. Here we report the identification of a multifunctional sorting protein, PACS-2, that integrates ER-mitochondria communication, ER homeostasis, and apoptosis. PACS-2 controls the apposition of mitochondria with the ER, as depletion of PACS-2 causes BAP31-dependent mitochondria fragmentation and uncoupling from the ER. PACS-2 also controls formation of ER lipid-synthesizing centers found on mitochondria-associated membranes and ER homeostasis. However, in response to apoptotic inducers, PACS-2 translocates Bid to mitochondria, which initiates a sequence of events including the formation of mitochondrial truncated Bid, the release of cytochrome c, and the activation of caspase-3, thereby causing cell death. Together, our results identify PACS-2 as a novel sorting protein that links the ER-mitochondria axis to ER homeostasis and the control of cell fate, and provide new insights into Bid action.

We report the role of one member of a novel gene family, PACS-1, in the localization of trans-Golgi network (TGN) membrane proteins. PACS-1 directs the TGN localization of furin by binding to the protease's phosphorylated cytosolic domain. Antisense studies show TGN localization of furin and mannose-6-phosphate receptor, but not TGN46, is strictly dependent on PACS-1. Analyses in vitro and in vivo show PACS-1 has properties of a coat protein and connects furin to components of the clathrin-sorting machinery. Cell-free assays indicate TGN localization of furin is directed by a PACS-1-mediated retrieval step. Together, these findings explain a mechanism by which membrane proteins in mammalian cells are localized to the TGN.

Copy number mutations implicate excess production of α-synuclein as a possibly causative factor in Parkinson’s disease (PD). Using an unbiased screen targeting endogenous gene expression, we discovered that the β2-adrenoreceptor (β2AR) is a regulator of the α-synuclein gene (SNCA). β2AR ligands modulate SNCA transcription through histone 3 lysine 27 acetylation of its promoter and enhancers. Over 11 years of follow-up in 4 million Norwegians, the β2AR agonist salbutamol, a brain-penetrant asthma medication, was associated with reduced risk of developing PD (rate ratio, 0.66; 95% confidence interval, 0.58 to 0.76). Conversely, a β2AR antagonist correlated with increased risk. β2AR activation protected model mice and patient-derived cells. Thus, β2AR is linked to transcription of α-synuclein and risk of PD in a ligand-specific fashion and constitutes a potential target for therapies.

The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor ␣ 1 -antitrypsin Portland (␣ 1 -PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed Hisand FLAG-tagged ␣ 1 -PDX (␣ 1 -PDX͞hf) revealed the selectivity of the ␣ 1 -PDX͞hf reactive site loop for furin (K i , 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that ␣ 1 -PDX͞hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin's active site, ␣ 1 -PDX͞hf partitions with equal probability to undergo proteolysis by furin at the Cterminal side of the reactive center -Arg 355 -Ile-Pro-Arg 358 -2 or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of ␣ 1 -PDX͞hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the ␣ 1 -PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.

Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to ␤ 1 -adrenergic receptor (␤ 1 AR)-selective activation 3.7-fold, to ␤ 2 AR-selective activation only 1.6-fold and to prostaglandin E 2 (PGE 2 ) not at all. Therefore, the rank order of efficacy in coupling to AC6 is ␤ 1 AR > ␤ 2 AR > prostaglandin E 2 receptor (EP 2 R). ␤ 2 AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing ␤ARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, ␤ 1 AR, and ␤ 2 AR, but not EP 2 R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by ␤AR subtypes but lack thereof by PGE 2 . When cardiomyocytes were stimulated with a ␤AR agonist, ␤ 2 AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of ␤ARKct. Thus, agonist-induced translocation of ␤ 2 AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of ␤ 2 AR coupling to AC6 as compared with ␤ 1 AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors.

contributed equally to this work PACS-1 is a cytosolic protein involved in controlling the correct subcellular localization of integral membrane proteins that contain acidic cluster sorting motifs, such as furin and human immunode®ciency virus type 1 (HIV-1) Nef. We have now investigated the interaction of PACS-1 with heterotetrameric adaptor complexes. PACS-1 associates with both AP-1 and AP-3, but not AP-2, and forms a ternary complex between furin and AP-1. A short sequence within PACS-1 that is essential for binding to AP-1 has been identi®ed. Mutation of this motif yielded a dominantnegative PACS-1 molecule that can still bind to acidic cluster motifs on cargo proteins but not to adaptor complexes. Expression of dominant-negative PACS-1 causes a mislocalization of both furin and mannose 6-phosphate receptor from the trans-Golgi network, but has no effect on the localization of proteins that do not contain acidic cluster sorting motifs. Furthermore, expression of dominant-negative PACS-1 inhibits the ability of HIV-1 Nef to downregulate MHC-I. These studies demonstrate the requirement for PACS-1 interactions with adaptor proteins in multiple processes, including secretory granule biogenesis and HIV-1 pathogenesis.

There is a growing body of evidence that G proteincoupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that ␤ 1 -and ␤ 2 -adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from ␤ 1 AR and ␤ 2 AR knockout mice, we found that stimulation of the ␤ 1 AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the ␤ 2 AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the ␤ 2 AR but not the ␤ 1 AR in neonatal cardiac myocytes. Evidence for ␤ 2 AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the ␤ 2 AR and caveolin 3, and co-migration of the ␤ 2 AR with a caveolin-3-enriched membrane fraction. The ␤ 2 AR-stimulated increase in the myocyte contraction rate is increased by ϳ2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of ␤ 2 AR and caveolin 3 and comigration of ␤ 2 AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on ␤ 1 AR signaling. These observations suggest that ␤ 2 ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.Catecholamines act through cardiac ␤-adrenergic receptors (␤ARs) 1 to modulate heart rate and contractility. Three ␤AR subtypes have been cloned (␤ 1 AR, ␤ 2 AR, and ␤ 3 AR). ␤ 1 AR and ␤ 2 AR are the primary subtypes responsible for cardiac response to catecholamines. ␤ 1 AR and ␤ 2 AR are also pharmacologically more similar to each other than they are to the ␤ 3 AR. The close structural and functional properties of ␤ 1 AR and ␤ 2 AR are paradigmatic of many other G protein-coupled receptor families in which two or more receptor subtypes respond to the same hormone or neurotransmitter and couple to the same effector systems. Although ␤ 1 AR and ␤ 2 AR have very similar signaling properties when expressed in undifferentiated cell lines (1), there is a growing body of experimental evidence that suggests that they have different signaling properties in regulating cardiac function. The ␤ 1 AR knockout (␤ 1 AR-KO) mice lack the normal chronotropic and inotropic responses to the non-selective agonist isoproterenol (2). Thus, in the murine heart, ␤ 2 ARs play no significant role in controlling heart rate and contractility. ␤ 2 AR knockout (␤ 2 AR-KO) mice have normal inotropic and chr...