Normal cellular function is hinged upon the ability of the endogenous machinery to properly process newly synthesized proteins, and this is often required for enabling their functional activity. Eukaryotic cells contain several proteinfolding, or chaperone, systems assisting in this process. Despite the apparent redundancy, it is believed that each chaperone system plays an important and unique role in facilitating protein folding by acting on distinct sets of substrates at unique cellular locations and/or under certain conditions (1, 2). One such chaperone system, unique to eukaryotic cells, is the Chaperonin Containing TCP-1 (CCT), 1 also known as TCP-1 Ring Complex (TRiC) (for reviews see 3, 1). CCT is composed of eight different subunits that assemble into a double ring structure, creating an internal cavity that serves as a folding chamber. A pioneering study demonstrated refolding of phytochrome, a light-sensing protein of plants, by a cytosolic molecular chaperone related to CCT (4). Studies in yeast and model mammalian cell lines indicate that the action of the CCT chaperonin is critical for cellular function. It is estimated that CCT may assist folding and assembly of up to 10% of all cellular proteins (5). Several recent studies reported identification of CCT substrates by proteomic and genetic methods (6, 7). Despite the important advances, these identified substrates are rather limited in number, and the set of the proteins requiring CCT assistance is likely to vary substantially across different specialized cells. Furthermore, virtually nothing is known about the involvement of the CCT chaperonin system in the regulation of specific cellular processes in the in vivo setting of complex multicellular organisms.Recent studies have established that the CCT function is regulated by phosducin-like proteins (PhLP) that are increasingly viewed as CCT co-chaperones (8). The best studied member of this family, PhLP, has been shown to be indispensable for the folding of the ␤ subunits of heterotrimeric G proteins that share a common WD40 motif with many CCT substrates (see ref. in 9). PhLP forms stable stoichiometric

Phosducin is a major phosphoprotein of rod photoreceptors that interacts with the G␤␥ 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. Phosducin (Pdc)2 was originally identified in the retina as an abundant 33-kDa cytosolic phosphoprotein phosphorylated in the dark and dephosphorylated in the light (1). Within the retina Pdc is expressed in both rod and cone photoreceptors (2, 3). The most distinguished feature of Pdc is its ability to form a specific complex with the ␤␥ subunits of visual heterotrimeric G protein, transducin (4, 5), and other heterotrimeric G proteins (6 -8). Affinity of Pdc toward G␤␥ is down-regulated by multiple phosphorylation and probably consequent binding of 14-3-3 protein (9 -10). Although the identity of Pdc kinase and phosphatase in photoreceptors remains unknown, the analysis of Pdc phosphorylation in vitro and ex vivo revealed that Pdc possesses multiple cAMP-dependent protein kinase and Ca 2ϩ / calmodulin-dependent protein kinase II phosphorylation sites (10 -12) and identified protein phosphatase 2A (PP2A) as the putative Pdc phosphatase (13).Despite the obvious progress in understanding the molecular basis of Pdc/G␤␥ interactions, the role of Pdc and its light-dependent phosphorylation in photoreceptors is poorly understood. Originally it was proposed that, upon activation by light, Pdc scavenges transducin ␤␥ subunits from phototransduction and by doing so reduces photoreceptor light s...

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 ␣ and ␤␥ functional units are subject to the regulation. Significance: We describe a potential mechanism for regulating the cellular levels of G proteins.

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.

Deregulation of cellular proteostasis due to the failure of the ubiquitin proteasome system to dispose of misfolded aggregation-prone proteins is a hallmark of various neurodegenerative diseases in humans. Microorganisms have evolved to survive massive protein misfolding and aggregation triggered by heat shock using their protein-unfolding ATPases (unfoldases) from the Hsp100 family. Because the Hsp100 chaperones are absent in homoeothermic mammals, we hypothesized that the vulnerability of mammalian neurons to misfolded proteins could be mitigated by expressing a xenogeneic unfoldase. To test this idea, we expressed proteasome-activating nucleotidase (PAN), a protein-unfolding ATPase from thermophilic , which is homologous to the 19S eukaryotic proteasome and similar to the Hsp100 family chaperones in rod photoreceptors of mice. We found that PAN had no obvious effect in healthy rods; however, it effectively counteracted protein-misfolding retinopathy in Gγ knock-out mice. We conclude that archaeal PAN can rescue a protein-misfolding neurodegenerative disease, likely by recognizing misfolded mammalian proteins. This study demonstrates successful therapeutic application of an archaeal molecular chaperone in an animal model of neurodegenerative disease. Introducing the archaeal protein-unfolding ATPase proteasome-activating nucleotidase (PAN) into the retinal photoreceptors of mice protected these neurons from the cytotoxic effect of misfolded proteins. We propose that xenogeneic protein-unfolding chaperones could be equally effective against other types of neurodegenerative diseases of protein-misfolding etiology.