The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C.

The target of rapamycin (TOR), as part of the rapamycin-sensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl-terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination-mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.

SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.

Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. Recent biochemical studies suggested that TORC2 is the elusive PDK2 for Akt/PKB Ser473 phosphorylation in the hydrophobic motif. Phosphorylation at Ser473, along with Thr308 of its activation loop, is deemed necessary for Akt function, although the regulatory mechanisms and physiological importance of each phosphorylation site remain to be fully understood. Here, we report that SIN1/MIP1 is an essential TORC2/PDK2 subunit. Genetic ablation of sin1 abolished Akt-Ser473 phosphorylation and disrupted rictor-mTOR interaction but maintained Thr308 phosphorylation. Surprisingly, defective Ser473 phosphorylation affected only a subset of Akt targets in vivo, including FoxO1/3a, while other Akt targets, TSC2 and GSK3, and the TORC1 effectors, S6K and 4E-BP1, were unaffected. Our findings reveal that the SIN1-rictor-mTOR function in Akt-Ser473 phosphorylation is required for TORC2 function in cell survival but is dispensable for TORC1 function.

Adaptations to an extreme environment: retinal organisation and spectral properties of photoreceptors in Antarctic notothenioid fish.

The Notothenioid suborder of teleosts comprises a number of species that live below the sea ice of the Antarctic. The presence of 'antifreeze' glycoproteins in these fish as an adaptation to freezing temperature has been well documented but little is known about the adaptations of the visual system of these fish to a light environment in which both the quantity and spectral composition of downwelling sunlight has been reduced by passage through ice and snow. In this study, we show that the red/long-wave sensitive (LWS) opsin gene is not present in these fish but a UV-sensitive short-wave sensitive (SWS1) pigment is expressed along with blue-sensitive (SWS2) and green/middle-wave sensitive (Rh2) pigments. The identity and spectral location of maximal absorbance of the SWS1 and Rh2 pigments was confirmed by in vitro expression of the recombinant opsins followed by regeneration with 11-cis retinal. Only the SWS2 pigment showed interspecific variations in peak absorbance. Expression of the Rh2 opsin is localised to double cone receptors in both the central and peripheral retina, whereas SWS2 opsin expression is present only in the peripheral retina. SWS1 cones could not be identified by either microspectrophotometry or in situ hybridisation, presumably reflecting their low number and/or uneven distribution across the retina. A study of photoreceptor organisation in the retina of two species, the shallower dwelling Trematomus hansoni and the deeper dwelling Dissostichus mawsoni, identified a square mosaic in the former, and a row mosaic in the latter species; the row mosaic in Dissostichus mawsoni with less tightly packed cone photoreceptors allows for a higher rod photoreceptor density.

Protein translocation across the endoplasmic reticulum membrane in cold-adapted organisms.

Secretory proteins enter the secretory pathway by translocation across the membrane of the endoplasmic reticulum (ER) via a channel formed primarily by the Sec61 protein. Protein translocation is highly temperature dependent in mesophilic organisms. We asked whether the protein translocation machinery of organisms from extremely cold habitats was adapted to function at low temperature and found that post-translational protein import into ER-derived microsomes from Antarctic yeast at low temperature was indeed more efficient than into mesophilic yeast microsomes. Analysis of the amino-acid sequences of the core component of the protein translocation channel, Sec61p, from Antarctic yeast species did not reveal amino-acid changes potentially adaptive for function in the cold, because the sequences were too divergent. We therefore analyzed Sec61alpha (vertebrate Sec61p) sequences and protein translocation into the ER of Antarctic and Arctic fishes and compared them to Sec61alpha and protein translocation into the ER of temperate-water fishes and mammals. Overall, Sec61alpha is highly conserved amongst these divergent taxa; a number of amino-acid changes specific to fishes are evident throughout the protein, and, in addition, changes specific to cold-water fishes cluster in the lumenal loop between transmembrane domains 7 and 8 of Sec61alpha, which is known to be important for protein translocation across the ER membrane. Secretory proteins translocated more efficiently into fish microsomes than into mammalian microsomes at 10 degrees C and 0 degrees C. The efficiency of protein translocation at 0 degrees C was highest for microsomes from a cold-water fish. Despite substantial differences in ER membrane lipid composition, ER membrane fluidity was identical in Antarctic fishes, mesophilic fishes and warm-blooded vertebrates, suggesting that membrane fluidity, although typically important for the function of the transmembrane proteins, is not limiting for protein translocation across the ER membrane in the cold. Collectively, our data suggest that the limited amino-acid changes in Sec61alpha from fishes may be functionally significant and represent adaptive changes that enhance channel function in the cold.