Marycharmain Belcastro

Disruption of the Chaperonin Containing TCP-1 Function Affects Protein Networks Essential for Rod Outer Segment Morphogenesis and Survival

Type II Chaperonin Containing TCP-1 (CCT, also known as TCP-1 Ring Complex, TRiC) is a multi-subunit molecular machine thought to assist in the folding of ∼10% of newly translated cytosolic proteins in eukaryotes. A number of proteins folded by CCT have been identified in yeast and cultured mammalian cells, however, the function of this chaperonin in vivo has never been addressed. Here we demonstrate that suppressing the CCT activity in mouse photoreceptors by transgenic expression of a dominant-negative mutant of the CCT cofactor, phosducin-like protein (PhLP), results in the malformation of the outer segment, a cellular compartment responsible for light detection, and triggers rapid retinal degeneration. Investigation of the underlying causes by quantitative proteomics identified distinct protein networks, encompassing ∼200 proteins, which were significantly affected by the chaperonin deficiency. Notably among those were several essential proteins crucially engaged in structural support and visual signaling of the outer segment such as peripherin 2, Rom1, rhodopsin, transducin, and PDE6. These data for the first time demonstrate that normal CCT function is ultimately required for the morphogenesis and survival of sensory neurons of the retina, and suggest the chaperonin CCT deficiency as a potential, yet unexplored, cause of neurodegenerative diseases.

Compartment-specific Phosphorylation of Phosducin in Rods Underlies Adaptation to Various Levels of Illumination

Phosducin is a major phosphoprotein of rod photoreceptors that interacts with the Gβγ subunits of heterotrimeric G proteins in its dephosphorylated state. Light promotes dephosphorylation of phosducin; thus, it was proposed that phosducin plays a role in the light adaptation of G protein-mediated visual signaling. Different functions, such as regulation of protein levels and subcellular localization of heterotrimeric G proteins, transcriptional regulation, and modulation of synaptic transmission have also been proposed. Although the molecular basis of phosducin interaction with G proteins is well understood, the physiological significance of light-dependent phosphorylation of phosducin remains largely hypothetical. In this study we quantitatively analyzed light dependence, time course, and subcellular localization of two principal light-regulated phosphorylation sites of phosducin, serine 54 and 71. To obtain physiologically relevant data, our experimental model exploited free-running mice and rats subjected to controlled illumination. We found that in the dark-adapted rods, phosducin phosphorylated at serine 54 is compartmentalized predominantly in the ellipsoid and outer segment compartments. In contrast, phosducin phosphorylated at serine 71 is present in all cellular compartments. The degree of phosducin phosphorylation in the dark appeared to be less than 40%. Dim light within rod operational range triggers massive reversible dephosphorylation of both sites, whereas saturating light dramatically increases phosphorylation of serine 71 in rod outer segment. These results support the role of phosducin in regulating signaling in the rod outer segment compartment and suggest distinct functions for phosphorylation sites 54 and 71.

Expression and subcellular distribution of UNC119a, a protein partner of transducin α subunit in rod photoreceptors

A recently discovered interaction of rod transducin α subunit (Gα(t1)) with UNC119a is thought to be important for transducin trafficking in photoreceptors. In this study, we analyzed the subcellular distribution of UNC119a under different conditions of illumination in vivo. Analyses by immunofluorescence and Western blotting of retina serial tangential sections demonstrated that UNC119a resides predominantly in the rod inner segment, with a small fraction of UNC119a also appearing to infiltrate the rod outer segment. Such a distribution is consistent with the proposed role of UNC119a in facilitating transducin transport from the rod inner segment to the outer segment in the dark. In addition, UNC119a was present in smaller amounts in the cell body and synaptic region of rods. The profile of UNC119a subcellular distribution remained largely unchanged under all tested conditions of illumination, and correlated with the profile of Gα(t1) following its light-dependent translocation. Quantification by Western blotting suggested that mouse retina contains ~17 pmol of UNC119a, giving a ~1 to 4 molar ratio of UNC119a to Gα(t1). Hence, light-translocated Gα(t1) can serve as a major partner of UNC119a. Supporting this role, the levels of UNC119a were downregulated by about 2-fold in mouse retina lacking Gα(t1). As a dominant partner, Gα(t1) may potentially modulate the function of other known UNC119a-interacting proteins involved in photoreceptor ciliary trafficking and synaptic regulation, in a light-dependent manner.

Phosphorylation of Phosducin Accelerates Rod Recovery from Transducin Translocation

PURPOSE In rods saturated by light, the G protein transducin undergoes translocation from the outer segment compartment, which results in the uncoupling of transducin from its innate receptor, rhodopsin. We measured the kinetics of recovery from this adaptive cellular response, while also investigating the role of phosducin, a phosphoprotein binding transducin βγ subunits in its de-phosphorylated state, in regulating this process. METHODS Mice were exposed to a moderate rod-saturating light triggering transducin translocation, and then allowed to recover in the dark while free running. The kinetics of the return of the transducin subunits to the outer segments were compared in transgenic mouse models expressing full-length phosducin, and phosducin lacking phosphorylation sites serine 54 and 71, using Western blot analysis of serial tangential sections of the retina. RESULTS In mice expressing normal phosducin, transducin α and βγ subunits returned to the outer segments with a half-time (t(1/2)) of ∼24 and 29 minutes, respectively. In the phosducin phosphorylation mutants, the transducin α subunit moved four times slower, with t(1/2) ∼95 minutes, while the movement of transducin βγ was less affected. CONCLUSIONS We demonstrate that the recovery of rod photoreceptors from the ambient saturating levels of illumination, in terms of the return of the light-dispersed transducin subunits to the rod outer segments, occurs six times faster than reported previously. Our data also support the notion that the accumulation of transducin α subunit in the outer segment is driven by its re-binding to the transducin βγ dimer, because this process is accelerated significantly by phosducin phosphorylation.

Essential Role of the Chaperonin CCT in Rod Outer Segment Biogenesis

PURPOSE While some evidence suggests an essential role for the chaperonin containing t-complex protein 1 (CCT) in ciliogenesis, this function remains poorly understood mechanistically. We used transgenic mice, previously generated in our lab, and characterized by a genetically-induced suppression of CCT in rod photoreceptors as well as a malformation of the rod sensory cilia, the outer segments, to gain new insights into this underlying molecular mechanism. METHODS The CCT activity in rod photoreceptors of mice was suppressed by overexpressing the chaperonin inhibitor, phosducin-like protein short, and the ensuing changes of cellular morphology were analyzed by light and electron microscopy. Protein expression levels were studied by fluorescent microscopy and Western blotting. RESULTS Suppressing the chaperonin made the photoreceptors incompetent to build their outer segments. Specifically, the CCT-deficient rods appeared unable to expand the outer segment plasma membrane, and accommodate growth of this compartment. Seeking the molecular mechanisms underlying such a shortcoming, we found that the affected rods could not express normal levels of Bardet-Biedl Syndrome (BBS) proteins 2, 5, and 7 and, owing to that deficiency, were unable to assemble the BBSome, a multisubunit complex responsible for ciliary trafficking. A similar effect in response to the chaperonin suppression was also observed in cultured ciliated cells. CONCLUSIONS Our data provide new evidence indicating the essential role of the chaperonin CCT in the biogenesis of vertebrate photoreceptor sensory cilia, and suggest that it may be due to the direct participation of the chaperonin in the posttranslational processing of selected BBS proteins and assembly of the BBSome.

Splice Isoforms of Phosducin-like Protein Control the Expression of Heterotrimeric G Proteins

Background: Heterotrimeric G proteins are essential for biological signaling; however, the mechanism of their biosynthesis remains poorly understood. Results: Long and short splice isoforms of phosducin-like protein stimulate and inhibit production of G proteins in the cell. Conclusion: Both G protein α and βγ functional units are subject to the regulation. Significance: We describe a potential mechanism for regulating the cellular levels of G proteins. Heterotrimeric G proteins play an essential role in cellular signaling; however, the mechanism regulating their synthesis and assembly remains poorly understood. A line of evidence indicates that the posttranslational processing of G protein β subunits begins inside the protein-folding chamber of the chaperonin containing t-complex protein 1. This process is facilitated by the ubiquitously expressed phosducin-like protein (PhLP), which is thought to act as a CCT co-factor. Here we demonstrate that alternative splicing of the PhLP gene gives rise to a transcript encoding a truncated, short protein (PhLPs) that is broadly expressed in human tissues but absent in mice. Seeking to elucidate the function of PhLPs, we expressed this protein in the rod photoreceptors of mice and found that this manipulation caused a dramatic translational and posttranslational suppression of rod heterotrimeric G proteins. The investigation of the underlying mechanism revealed that PhLPs disrupts the folding of Gβ and the assembly of Gβ and Gγ subunits, events normally assisted by PhLP, by forming a stable and apparently inactive tertiary complex with CCT preloaded with nascent Gβ. As a result, the cellular levels of Gβ and Gγ, which depends on Gβ for stability, decline. In addition, PhLPs evokes a profound and rather specific down-regulation of the Gα transcript, leading to a complete disappearance of the protein. This study provides the first evidence of a generic mechanism, whereby the splicing of the PhLP gene could potentially and efficiently regulate the cellular levels of heterotrimeric G proteins.

Farnesylation of the Transducin G Protein Gamma Subunit Is a Prerequisite for Its Ciliary Targeting in Rod Photoreceptors

Primary cilia are microtubule-based organelles, which protrude from the plasma membrane and receive a wide range of extracellular signals. Various cilia use G protein-coupled receptors (GPCRs) for the detection of these signals. For instance, vertebrate rod photoreceptors use their cilia (also called outer segments) as antennae detecting photons by GPCR rhodopsin. Rhodopsin recognizes incoming light and activates its G protein, transducin, which is composed of three subunits α, β, and γ. Similar to all G protein γ subunits, the transducin Gγ1 subunit undergoes C-terminal prenylation resulting in the addition of an isoprenoid farnesyl; however, the significance of this posttranslational modification is unclear. To study the role of the farnesyl group, we genetically introduced a mutant Gγ1 that lacked the prenylation site into the retinal photoreceptors of mice. The biochemical and physiological analyses of these mice revealed that mutant Gγ1 dimerizes with the endogenous transducin Gβ1 subunit and that the resulting Gβγ dimers display reduced hydrophobicity. Although mutant Gβγ dimers could form a heterotrimeric G protein, they could not mediate phototransduction. This deficiency was due to a strong exclusion of non-farnesylated Gβγ complexes from the cilia (rod outer segments). Our results provide the first evidence that farnesylation is required for trafficking of G-protein βγ subunits to the cilium of rod photoreceptors.