RESUMO
In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , Proteínas de Membrana/química , Microssomos/metabolismo , Dados de Sequência Molecular , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transporte Proteico , Canais de Translocação SEC , Lobos/metabolismoRESUMO
Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co- and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.
Assuntos
DNA Helicases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/metabolismo , Animais , DNA Helicases/genética , Retículo Endoplasmático/genética , Inativação Gênica , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Chaperonas Moleculares , Células NIH 3T3 , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas de Ligação a RNA , Canais de Translocação SECRESUMO
The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Cromossomos Humanos Par 3/genética , Amplificação de Genes/genética , Neoplasias Pulmonares/genética , Proteínas de Membrana Transportadoras/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Inibidores Enzimáticos/farmacologia , Inativação Gênica/fisiologia , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Tapsigargina/farmacologia , Neoplasias da Glândula Tireoide/genéticaRESUMO
Ribosomes synthesizing secretory and membrane proteins are bound to the endoplasmic reticulum (ER) membrane and attach to ribosome-associated membrane proteins such as the Sec61 complex, which forms the protein-conducting channel in the membrane. The ER membrane-resident Hsp40 protein ERj1 was characterized as being able to recruit BiP to ribosomes in solution and to regulate protein synthesis in a BiP-dependent manner. Here, we show that ERj1 and Sec61 are associated with ribosomes at the ER of human cells and that the binding of ERj1 to ribosomes occurs with a binding constant in the picomolar range and is prevented by pretreatment of ribosomes with RNase. However, the affinity of ERj1 for ribosomes dramatically changes upon binding of BiP. This modulation by BiP may be responsible for the dual role of ERj1 at the ribosome, i.e. acting as a recruiting factor for BiP and regulating translation.