ER Targeting Signals: More than Meets the Eye?

The signal sequences that target newly synthesized proteins to the endoplasmic reticulum are highly variable; however, the functional significance of this diversity has remained obscure. In this issue, Kang et al. (2006) report that variability in signal sequences allows the cell to selectively regulate the translocation of proteins into the endoplasmic reticulum in a substrate-specific manner

Journal Staff

Inhibitors of the catalytic activity of the 20S proteasome are cytotoxic to tumor cells and are currently in clinical use for treatment of multiple myeloma, whilst the deubiquitinase activity associated with the 19S regulatory subunit of the proteasome is also a valid target for anti-cancer drugs. The mechanisms underlying the therapeutic efficacy of these drugs and their selective toxicity towards cancer cells are not known. Here, we show that increasing the cellular levels of proteasome substrates using an inhibitor of Sec61-mediated protein translocation significantly increases the extent of apoptosis that is induced by inhibition of proteasomal deubiquitinase activity in both cancer derived and non-transformed cell lines. Our results suggest that increased generation of misfolded proteasome substrates may contribute to the mechanism(s) underlying the increased sensitivity of tumor cells to inhibitors of the ubiquitin-proteasome system

Restoration of mutant bestrophin-1 expression, localisation and function in a polarised epithelial cell model

Autosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca2+-gated Cl− channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl− ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl− conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1

Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response

Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. ESI (eeyarestatin I) is a novel modulator of ER (endoplasmic reticulum) function. In the present study, we show that in addition to acutely inhibiting ERAD (ER-associated degradation), ESI causes production of mislocalized polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest that these non-translocated polypeptides promote activation of the UPR (unfolded protein response), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These results suggest that the accumulation of non-translocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation

Glycerolipid synthesis in heart cells

Isolated ventricular myocytes were used to study the glycerolipid synthesis process in the rat heart. The presence of the following lipogenic enzymes: fatty acyl-CoA synthetase (FAS), microsomal and mitochondrial forms of glycerolphosphate acyltransferase (GPAT), monoacylglycerolphosphate acyltransferase (MGPAT), phosphatidate phosphohydrolase (PAP)-1 and -2, and diacylglycerol acyltransferase (DGAT); was demonstrated in homogenates of ventricle muscle and cardiac myocytes. The specific activities of these enzymes were generally similar in the two preparations, with the exception of PAP-1 and -2 which were significantly lower in myocytes than in ventricle muscle. Further studies were undertaken to elucidate the nature of GPAT and PAP in ventricle muscle and compared with those of the more extensively characterised adipose tissue and liver enzymes. GPAT activity in the ventricle sarcoplasmic reticulum fraction had a 4-fold higher Km for glycerol-3-phosphate than GPAT in adipose tissue microsomes. Translocation of PAP-1 from a ventricle membrane fraction was demonstrated in response to lowering ionic strength. Incubation of myocytes with palmitate promoted translocation of PAP to membranes. PAP-2 in ventricle homogenates and membrane fractions was inhibited by physiological concentrations of Mg2+. A radioisotope pulse-chase protocol, established for use in perfused hearts, was adapted to measure glycerolipid turnover in isolated Ca2+-tolerant myocytes. The cells actively synthesised triacylglycerol (TAG) and phospholipids, and turned over their endogenous TAG pool so that oxidation of endogenous fatty acids occurred. Adrenaline increased TAG and phospholipid synthesis, stimulated lipolysis and endogenous fatty acid oxidation, but decreased oxidation of exogenous fatty acids. Adrenaline also stimulated microsomal GPAT activity and decreased association of PAP with myocyte membranes. Both α1- and β-adrenoceptor-mediated pathways appeared to be necessary for complete adrenergic stimulation of TAG synthesis, whilst α1-adrenergic stimulation was sufficient to fully mimic the inhibition of exogenous fatty acid oxidation. Insulin stimulated TAG but not phospholipid synthesis. Neither basal nor adrenaline-stimulated rates of lipolysis were inhibited by insulin. Increasing the concentration exogenous fatty acids enhanced utilisation of these substrates by myocytes, but suppressed the mobilisation and oxidation of endogenous fatty acids. The effects of adrenaline were apparent at all exogenous fatty acid concentrations examined

Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein

Missense mutations in human PLP1, the gene encoding myelin proteolipid protein (PLP), cause dysmyelinating Pelizaeus-Merzbacher disease of varying severity. Although disease pathology has been linked to retention of misfolded PLP in the endoplasmic reticulum (ER) and induction of the unfolded protein response (UPR), the molecular mechanisms that govern phenotypic heterogeneity remain poorly understood. To address this issue, we examined the cellular response to missense mutants of PLP that are associated with distinct disease phenotypes. We found that the mild-disease-associated mutants, W162L and G245A, were cleared from the ER comparatively quickly via proteasomal degradation and/or ER exit. By contrast, the more `aggressive' A242V mutant, which causes severe disease, was significantly more stable, accumulated at the ER and resulted in a specific activation of the UPR. On the basis of these findings, we propose that the rate at which mutant PLP proteins are cleared from the ER modulates disease severity by determining the extent to which the UPR is activated

Role of calnexin in the glycan-independent quality control of proteolipid protein

The endoplasmic (ER) quality control apparatus ensures that misfolded or unassembled proteins are not deployed within the cell, but are retained in the ER and degraded. A glycoprotein-specific system involving the ER lectins calnexin and calreticulin is well documented, but very little is known about mechanisms that may operate for non-glycosylated proteins. We have used a folding mutant of a non- glycosylated membrane protein, proteolipid protein (PLP), to examine the quality control of this class of polypeptide. We find that calnexin associates with newly synthesized PLP molecules, binding stably to misfolded PLP. Calnexin also binds stably to an isolated transmembrane domain of PLP, suggesting that this chaperone is able to monitor the folding and assembly of domains within the ER membrane. Notably, this glycan-independent interaction with calnexin significantly retards the degradation of misfolded PLP. We propose that calnexin contributes to the quality control of non-glycosylated polytopic membrane proteins by binding to misfolded or unassembled transmembrane domains, and discuss our findings in relation to the role of calnexin in the degradation of misfolded proteins