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.
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.
Sato
M
M
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan.
Sato
K
K
Nakano
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004271:DNA, Fungal; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D008358:Mannose; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
D004271:DNA, Fungal; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; C089137:RER1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; C071239:SEC12 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C059010:dipeptidyl aminopeptidase B; D008358:Mannose