RESUMO
Calreticulin (CALR) is an endoplasmic reticulum (ER)-retained chaperone that assists glycoproteins in obtaining their structure. CALR mutations occur in patients with myeloproliferative neoplasms (MPNs), and the ER retention of CALR mutants (CALR MUT) is reduced due to a lacking KDEL sequence. Here, we investigate the impact of CALR mutations on protein structure and protein levels in MPNs by subjecting primary patient samples and CALR-mutated cell lines to limited proteolysis-coupled mass spectrometry (LiP-MS). Especially glycoproteins are differentially expressed and undergo profound structural alterations in granulocytes and cell lines with homozygous, but not with heterozygous, CALR mutations. Furthermore, homozygous CALR mutations and loss of CALR equally perturb glycoprotein integrity, suggesting that loss-of-function attributes of mutated CALR chaperones (CALR MUT) lead to glycoprotein maturation defects. Finally, by investigating the misfolding of the CALR glycoprotein client myeloperoxidase (MPO), we provide molecular proof of protein misfolding in the presence of homozygous CALR mutations.
Assuntos
Calreticulina , Transtornos Mieloproliferativos , Humanos , Calreticulina/genética , Calreticulina/química , Calreticulina/metabolismo , Mutação/genética , Homozigoto , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteoma/metabolismoRESUMO
Although prion infections cause cognitive impairment and neuronal death, transcriptional and translational profiling shows progressive derangement within glia but surprisingly little changes within neurons. Here we expressed PrPC selectively in neurons and astrocytes of mice. After prion infection, both astrocyte and neuron-restricted PrPC expression led to copious brain accumulation of PrPSc . As expected, neuron-restricted expression was associated with typical prion disease. However, mice with astrocyte-restricted PrPC expression experienced a normal life span, did not develop clinical disease, and did not show astro- or microgliosis. Besides confirming that PrPSc is innocuous to PrPC -deficient neurons, these results show that astrocyte-born PrPSc does not activate the extreme neuroinflammation that accompanies the onset of prion disease and precedes any molecular changes of neurons. This points to a nonautonomous mechanism by which prion-infected neurons instruct astrocytes and microglia to acquire a specific cellular state that, in turn, drives neural dysfunction.
Assuntos
Doenças Priônicas , Príons , Animais , Astrócitos/metabolismo , Camundongos , Neuroglia/metabolismo , Neurônios/metabolismo , Doenças Priônicas/metabolismo , Príons/metabolismoRESUMO
Brain-matter vacuolation is a defining trait of all prion diseases, yet its cause is unknown. Here, we report that prion infection and prion-mimetic antibodies deplete the phosphoinositide kinase PIKfyve-which controls endolysosomal maturation-from mouse brains, cultured cells, organotypic brain slices, and brains of Creutzfeldt-Jakob disease victims. We found that PIKfyve is acylated by the acyltransferases zDHHC9 and zDHHC21, whose juxtavesicular topology is disturbed by prion infection, resulting in PIKfyve deacylation and rapid degradation, as well as endolysosomal hypertrophy and activation of TFEB-dependent lysosomal enzymes. A protracted unfolded protein response (UPR), typical of prion diseases, also induced PIKfyve deacylation and degradation. Conversely, UPR antagonists restored PIKfyve levels in prion-infected cells. Overexpression of zDHHC9 and zDHHC21, administration of the antiprion polythiophene LIN5044, or supplementation with the PIKfyve reaction product PI(3,5)P2 suppressed prion-induced vacuolation and restored lysosomal homeostasis. Thus, PIKfyve emerges as a central mediator of vacuolation and neurotoxicity in prion diseases.
Assuntos
Fosfatidilinositol 3-Quinases , Doenças Priônicas , Aciltransferases , Animais , Encéfalo/metabolismo , Homeostase , Lisossomos/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as a potential therapeutic agent in treating peripheral demyelinating and dysmyelinating diseases. We designed a dimeric Fc-fusion protein comprising the myelinotrophic domain of PrP (FT2Fc), which activated Adgrg6 in vitro and exhibited favorable pharmacokinetic properties for in vivo treatment of peripheral neuropathies. While chronic FT2Fc treatment elicited specific transcriptomic changes in the sciatic nerves of PrP knockout mice, no amelioration of the early molecular signs demyelination was detected. Instead, RNA sequencing of sciatic nerves revealed downregulation of cytoskeletal and sarcomere genes, akin to the gene expression changes seen in myopathic skeletal muscle of PrP overexpressing mice. These results call for caution when devising myelinotrophic therapies based on PrP-derived Adgrg6 ligands. While our treatment approach was not successful, Adgrg6 remains an attractive therapeutic target to be addressed in other disease models or by using different biologically active Adgrg6 ligands.
Assuntos
Doenças Desmielinizantes/tratamento farmacológico , Fragmentos de Peptídeos/uso terapêutico , Proteínas Priônicas/química , Receptores Acoplados a Proteínas G/agonistas , Animais , Linhagem Celular , Doenças Desmielinizantes/genética , Feminino , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Proteínas Priônicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Nervo Isquiático/metabolismo , TranscriptomaRESUMO
Myelin is a multilamellar sheath generated by specialized glia called Schwann cells (SCs) in the peripheral nervous system (PNS), which serves to protect and insulate axons for rapid neuronal signaling. In zebrafish and rodent models, we identify GPR56/ADGRG1 as a conserved regulator of PNS development and health. We demonstrate that, during SC development, GPR56-dependent RhoA signaling promotes timely radial sorting of axons. In the mature PNS, GPR56 is localized to distinct SC cytoplasmic domains, is required to establish proper myelin thickness, and facilitates organization of the myelin sheath. Furthermore, we define plectin-a scaffolding protein previously linked to SC domain organization, myelin maintenance, and a series of disorders termed "plectinopathies"-as a novel interacting partner of GPR56. Finally, we show that Gpr56 mutants develop progressive neuropathy-like symptoms, suggesting an underlying mechanism for peripheral defects in some human patients with GPR56 mutations. In sum, we define Gpr56 as a new regulator in the development and maintenance of peripheral myelin.
Assuntos
Bainha de Mielina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Animais , Citoesqueleto/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Mutação/genética , Bainha de Mielina/ultraestrutura , Plectina/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Three decades after the discovery of prions as the cause of Creutzfeldt-Jakob disease and other transmissible spongiform encephalopathies, we are still nowhere close to finding an effective therapy. Numerous pharmacological interventions have attempted to target various stages of disease progression, yet none has significantly ameliorated the course of disease. We still lack a mechanistic understanding of how the prions damage the brain, and this situation results in a dearth of validated pharmacological targets. In this review, we discuss the attempts to interfere with the replication of prions and to enhance their clearance. We also trace some of the possibilities to identify novel targets that may arise with increasing insights into prion biology.
Assuntos
Doenças Priônicas/tratamento farmacológico , Príons/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , HumanosRESUMO
Prion infections cause inexorable, progressive neurological dysfunction and neurodegeneration. Expression of the cellular prion protein PrPC is required for toxicity, suggesting the existence of deleterious PrPC-dependent signaling cascades. Because group-I metabotropic glutamate receptors (mGluR1 and mGluR5) can form complexes with the cellular prion protein (PrPC), we investigated the impact of mGluR1 and mGluR5 inhibition on prion toxicity ex vivo and in vivo. We found that pharmacological inhibition of mGluR1 and mGluR5 antagonized dose-dependently the neurotoxicity triggered by prion infection and by prion-mimetic anti-PrPC antibodies in organotypic brain slices. Prion-mimetic antibodies increased mGluR5 clustering around dendritic spines, mimicking the toxicity of Aß oligomers. Oral treatment with the mGluR5 inhibitor, MPEP, delayed the onset of motor deficits and moderately prolonged survival of prion-infected mice. Although group-I mGluR inhibition was not curative, these results suggest that it may alleviate the neurological dysfunctions induced by prion diseases.
Assuntos
Proteínas PrPC/toxicidade , Doenças Priônicas/tratamento farmacológico , Doenças Priônicas/metabolismo , Piridinas/administração & dosagem , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Animais , Anticorpos/administração & dosagem , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Doenças Priônicas/genética , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Receptor de Glutamato Metabotrópico 5/genética , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismoRESUMO
Ablation of the cellular prion protein PrP(C) leads to a chronic demyelinating polyneuropathy affecting Schwann cells. Neuron-restricted expression of PrP(C) prevents the disease, suggesting that PrP(C) acts in trans through an unidentified Schwann cell receptor. Here we show that the cAMP concentration in sciatic nerves from PrP(C)-deficient mice is reduced, suggesting that PrP(C) acts via a G protein-coupled receptor (GPCR). The amino-terminal flexible tail (residues 23-120) of PrP(C) triggered a concentration-dependent increase in cAMP in primary Schwann cells, in the Schwann cell line SW10, and in HEK293T cells overexpressing the GPCR Adgrg6 (also known as Gpr126). By contrast, naive HEK293T cells and HEK293T cells expressing several other GPCRs did not react to the flexible tail, and ablation of Gpr126 from SW10 cells abolished the flexible tail-induced cAMP response. The flexible tail contains a polycationic cluster (KKRPKPG) similar to the GPRGKPG motif of the Gpr126 agonist type-IV collagen. A KKRPKPG-containing PrPC-derived peptide (FT(23-50)) sufficed to induce a Gpr126-dependent cAMP response in cells and mice, and improved myelination in hypomorphic gpr126 mutant zebrafish (Danio rerio). Substitution of the cationic residues with alanines abolished the biological activity of both FT(23-50) and the equivalent type-IV collagen peptide. We conclude that PrP(C) promotes myelin homeostasis through flexible tail-mediated Gpr126 agonism. As well as clarifying the physiological role of PrP(C), these observations are relevant to the pathogenesis of demyelinating polyneuropathies--common debilitating diseases for which there are limited therapeutic options.
Assuntos
Príons/metabolismo , Príons/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Colágeno Tipo IV/química , Colágeno Tipo IV/farmacologia , AMP Cíclico/metabolismo , Doenças Desmielinizantes/metabolismo , Feminino , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Bainha de Mielina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Maleabilidade , Proteínas Priônicas , Príons/química , Príons/genética , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
The pathogenesis of acquired myeloperoxidase (MPO) deficiency, a rare phenomenon observed in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), is unknown. MPO is a glycoprotein (GP) chaperoned by calreticulin (CALR) in the endoplasmic reticulum. Mutations in CALR are frequently found in patients with myelofibrosis (MF) and essential thrombocythemia (ET) with nonmutated Janus kinase 2 (JAK2). We hypothesized that acquired MPO deficiency in MPN could be associated with the presence of CALR mutations. A cohort of 317 patients with MPN (142 polycythemia vera [PV], 94 ET, and 81 MF) was screened for MPO deficiency. MPO deficiency was observed in 6/81 MF patients (7.4%), but not in PV or ET patients. Susceptibility to infections had been documented in 2/6 (33%) MPO-deficient patients. Five out of 6 patients with MPO deficiency carried a homozygous CALR mutation and were also deficient in eosinophilic peroxidase (EPX). In contrast, 1 patient with MF, a JAK2-V617F mutation, and MPO deficiency, carried 2 previously reported MPO mutations and showed normal EPX activity. Patients with homozygous CALR mutations had reduced MPO protein, but normal MPO messenger RNA (mRNA) levels supporting a posttranscriptional defect in MPO production. Finally, we demonstrate in vitro that in the absence of CALR, immature MPO protein precursors are degraded in the proteasome. Therefore, 4 decades after the first description of acquired MPO deficiency in MPN, we provide the molecular correlate associated with this phenomenon and evidence that CALR mutations can affect the biosynthesis of GPs.
Assuntos
Calreticulina/genética , Erros Inatos do Metabolismo/genética , Mutação , Mielofibrose Primária/genética , Animais , Células Cultivadas , Estudos de Coortes , Homozigoto , Humanos , Erros Inatos do Metabolismo/patologia , Camundongos , Camundongos Knockout , Peroxidase/genética , Peroxidase/metabolismo , Mielofibrose Primária/complicações , Mielofibrose Primária/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , ProteóliseRESUMO
The coalescence of proteins into highly ordered aggregates is a hallmark of protein misfolding disorders (PMDs), which, when affecting the central nervous system, lead to progressive neurodegeneration. Although the chemical identity and the topology of each culprit protein are unique, the principles governing aggregation and propagation are strikingly stereotypical. It is now clear that such protein aggregates can spread from cell to cell and eventually affect entire organ systems - similarly to prion diseases. However, because most aggregates are not found to transmit between individuals, they are not infectious sensu strictiori. Therefore, they are not identical to prions and we prefer to define them as 'prionoids'. Here we review recent advances in understanding the toxicity of protein aggregation affecting the brain.
Assuntos
Proteínas Priônicas/fisiologia , Peptídeos beta-Amiloides/fisiologia , Animais , Humanos , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Dobramento de Proteína , Multimerização Proteica , Transporte Proteico , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Proteínas tau/fisiologiaRESUMO
The cellular prion protein (PrPC) consists of a flexible N-terminal tail (FT, aa 23-128) hinged to a membrane-anchored globular domain (GD, aa 129-231). Ligation of the GD with antibodies induces rapid neurodegeneration, which is prevented by deletion or functional inactivation of the FT. Therefore, the FT is an allosteric effector of neurotoxicity. To explore its mechanism of action, we generated transgenic mice expressing the FT fused to a GPI anchor, but lacking the GD (PrPΔ141-225, or "FTgpi"). Here we report that FTgpi mice develop a progressive, inexorably lethal neurodegeneration morphologically and biochemically similar to that triggered by anti-GD antibodies. FTgpi was mostly retained in the endoplasmic reticulum, where it triggered a conspicuous unfolded protein response specifically activating the PERK pathway leading to phosphorylation of eIF2α and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection.
Assuntos
Cerebelo/patologia , Proteínas PrPC/metabolismo , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Resposta a Proteínas não Dobradas , Animais , Cerebelo/metabolismo , Estresse do Retículo Endoplasmático , Camundongos , Camundongos Transgênicos , Proteínas PrPC/análise , PríonsRESUMO
Calnexin is a well-characterized transmembrane chaperone involved in the folding of newly synthesized glycoproteins in the lumen of the endoplasmic reticulum (ER). Here, we reveal a previously unrecognized function of calnexin in regulating the transcriptional response downstream of epidermal growth factor receptor (EGF), the product of a well-known human oncogene. We find that cell stimulation with EGF leads to the caspase-8-dependent cleavage of the calnexin cytoplasmic domain, preferentially at ER-mitochondria interaction sites. The released fragment translocates into the nucleus, binds to PIAS3--a natural inhibitor of activated STAT3--and, thus, acts as an enhancer of the STAT3-mediated transcriptional response to EGF. Also, we reveal the unsuspected capacity of calnexin to sense ER stress and, in response, prevent the EGF-induced processing of its cytosolic domain. Thus, cells integrate the health status of the ER to determine the amplitude of their response to EGF.
Assuntos
Calnexina/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Receptores ErbB/metabolismo , Fator de Transcrição STAT3/metabolismo , Sequência de Aminoácidos , Calnexina/química , Caspase 8/metabolismo , Linhagem Celular , Receptores ErbB/genética , Humanos , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Transdução de Sinais , Transcrição GênicaRESUMO
Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120-160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte Proteico , Proteínas/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Trifosfato de Adenosina/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana Transportadoras/genética , Sinais Direcionadores de Proteínas/genética , Interferência de RNA , RNA Interferente Pequeno , Transdução de SinaisRESUMO
The signal recognition particle (SRP) is a ubiquitous cytoplasmic ribonucleoprotein complex required for the cotranslational targeting of proteins to the endoplasmic reticulum (ER). In eukaryotes, SRP has to arrest the elongation of the nascent chains during targeting to ensure efficient translocation of the preprotein, and this function of SRP is dependent on SRP9/14. Here we present the results of a mutational study on the human protein h9/14 that identified and characterized regions and single residues essential for elongation arrest activity. Effects of the mutations were assessed both in cell-free translation/translocation assays and in cultured mammalian cells. We identified two patches of basic amino acid residues that are essential for activity, whereas the internal loop of SRP14 was found to be dispensable. One patch of important basic residues comprises the previously identified basic pentapetide KRDKK, which can be substituted by four lysines without loss of function. The other patch includes three lysines in the solvent-accessible alpha2 of h9. All essential residues are located in proximity in SRP9/14 and their basic character suggests that they serve as a positively charged platform for interactions with ribosomal RNA. In addition, they can all be lysines consistent with the hypothesis that they recognize their target(s) via electrostatic contacts, most likely with the phosphate backbone, as opposed to contacts with specific bases.
Assuntos
Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Linhagem Celular , Sequência Conservada , Teste de Complementação Genética , Humanos , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Elongação Traducional da Cadeia Peptídica , Multimerização Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Partícula de Reconhecimento de Sinal/genética , Eletricidade EstáticaRESUMO
SRP is essential for targeting nascent chains to the endoplasmic reticulum, and it delays nascent chain elongation in cell-free translation systems. However, the significance of this function has remained unclear. We show that efficient protein translocation into the ER is incompatible with normal cellular translation rates due to rate-limiting concentrations of SRP receptor (SR). We complemented mammalian cells depleted of SRP14 by expressing mutant versions of the protein lacking the elongation arrest function. The absence of a delay caused inefficient targeting of preproteins leading to defects in secretion, depletion of proteins in the endogenous membranes, and reduced cell growth. The detrimental effects were reversed by either reducing the cellular protein synthesis rate or increasing SR expression. SRP therefore ensures that nascent chains remain translocation competent during the targeting time window dictated by SR. Since SRP-signal sequence affinities vary, the delay may also regulate which proteins are preferentially targeted.
Assuntos
Retículo Endoplasmático Rugoso/metabolismo , Elongação Traducional da Cadeia Peptídica , Partícula de Reconhecimento de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Ciclo Celular , Linhagem Celular , Sistema Livre de Células , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/genéticaRESUMO
The signal recognition particle (SRP) is required for protein translocation into the endoplasmic reticulum (ER). With RNA interference we reduced its level about ten-fold in mammalian cells to study its cellular functions. Such low levels proved insufficient for efficient ER-targeting, since the accumulation of several proteins in the secretory pathway was specifically diminished. Although the cells looked unaffected, they displayed noticeable and selective defects in post-ER membrane trafficking. Specifically, the anterograde transport of VSV-G and the retrograde transport of the Shiga toxin B-subunit were stalled at the level of the Golgi whereas the endocytosed transferrin receptor failed to recycle to the plasma membrane. Endocytic membrane trafficking from the plasma membrane to lysosomes or Golgi was undisturbed and major morphological changes in the ER and the Golgi were undetectable at low resolution. Selective membrane trafficking defects were specifically suppressed under conditions when low levels of SRP became sufficient for efficient ER-targeting and are therefore a direct consequence of the lower targeting capacity of cells with reduced SRP levels. Selective post-ER membrane trafficking defects occur at SRP levels sufficient for survival suggesting that changes in SRP levels and their effects on post-ER membrane trafficking might serve as a mechanism to alter temporarily the localization of selected proteins.