RESUMEN

In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding alpha-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated alpha-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca2+ ions, whereas a lack of alpha-COP expression was lethal. The alpha-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca2+-ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca2+ dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca2+ homeostasis is discussed.

Asunto(s)

Calcio/metabolismo , Proteína Coatómero/metabolismo , Pichia/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Western Blotting , ATPasas Transportadoras de Calcio/metabolismo , Clonación Molecular , Retículo Endoplásmico/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Humanos , Immunoblotting , Glicoproteínas de Membrana/metabolismo , Modelos Genéticos , Chaperonas Moleculares , Datos de Secuencia Molecular , Mutación , Fenotipo , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Proteínas/metabolismo , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo