Endoplasmic reticulum localization of Sec12p is achieved by two mechanisms: Rer1p-dependent retrieval that requires the transmembrane domain and Rer1p-independent retention that involves the cytoplasmic domain

• Published in: The Journal of cell biology Vol. 134; no. 2; pp. 279 - 293
• Main Authors: Sato, M. (University of Tokyo, Tokyo, Japan.), Sato, K, Nakano, A
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01.07.1996; The Rockefeller University Press
• Subjects: Amino Acid Sequence; amino acid sequences; Antibodies; APARATO GOLGI; APPAREIL DE GOLGI; Base Sequence; binding proteins; Binding Sites; cell membranes; Cells; Cellular biology; Chimeras; Cloning, Molecular; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Cytoplasm; Cytoplasm - metabolism; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - genetics; DNA, Fungal; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; fluorescent antibody technique; Fungal Proteins; Fungal Proteins - genetics; Fungal Proteins - metabolism; genetics; Golgi apparatus; Guanine Nucleotide Exchange Factors; immunocytochemistry; IMMUNOFLUORESCENCE; IMMUNOLOGIE; INMUNOFLUORESCENCIA; INMUNOLOGIA; Mannose; MEMBRANAS CELULARES; MEMBRANE CELLULAIRE; Membrane Glycoproteins; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Membrane Proteins; Membrane Proteins - metabolism; metabolism; METABOLISME DES PROTEINES; METABOLISMO PROTEICO; Molecular Sequence Data; Molecules; Mutation; Perceptual localization; protein secretion; Protein Sorting Signals; Protein Sorting Signals - metabolism; protein transport; PROTEINAS; PROTEINAS AGLUTINANTES; PROTEINE; PROTEINE DE LIAISON; Recombinant Fusion Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; RETICULO ENDOPLASMATICO; RETICULUM ENDOPLASMIQUE; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; SECRECION; SECRETION; transmembrane proteins; Vacuoles; Vesicular Transport Proteins; Yeast; Yeasts
• ISSN: 0021-9525; 1540-8140; 1540-8140
• DOI: 10.1083/jcb.134.2.279

Yeast Sec12p is a type II transmembrane protein in the ER, which is essential for the formation of transport vesicles. From biochemical and morphological lines of evidence, we have proposed that Sec12p is localized to the ER by two mechanisms: static retention in the ER and dynamic retrieval from the early Golgi compartment. We have also shown that Rer1p, a membrane protein in the Golgi, is required for correct localization of Sec12p. In the present study, we have performed a systematic analysis to determine the ER localization signals in Sec12p corresponding to these two mechanisms. Both the transmembrane domain (TMD) and the NH2-terminal cytoplasmic domain of Sec12p show the ability to localize the protein to the ER. The effect of the TMD is potent and sufficient by itself for the ER localization and is strongly dependent on Rer1p. On the other hand, the cytoplasmic domain shows a moderate ER-localization capability which is independent of Rer1p. The rate of mannosyl modification has been measured to distinguish between retention and retrieval. The cytoplasmic domain significantly delays the transport from the ER to the cis-Golgi. In contrast, the TMD shows only a subtle retardation in the transport from the ER to the cis-Golgi but strictly prevents the transport beyond there. From these observations, we conclude that the TMD mainly acts as the retrieval signal and the cytoplasmic domain contains the retention signal. This study not only supports the two-mechanisms hypothesis but also provides powerful tools to dissect the two