Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway.
ERGIC-53 is a lectin-type membrane protein that continuously recycles between the ER, ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. To identify the targeting signals that mediate this recycling, N-glycosylated and myc-tagged variants of ERGIC-53 were constructed. By monitoring endoglycosidase H resistance, we measured the loss from the ER-ERGIC-cis-Golgi cycle of ERGIC-53. A domain exchange approach with the plasma membrane reporter protein CD4 showed that the transmembrane and the lumenal domains are not sufficient, while the cytoplasmic domain of ERGIC-53 is required and sufficient for pre-medial-Golgi localization. However, the ERGIC-53 cytoplasmic domain on CD4 lead to increased ER-staining by immunofluorescence microscopy indicating that this domain alone cannot provide for unbiased recycling through the ER-ERGIC-cis-Golgi compartments. Complete progress through the ER-ERGIC-cis-Golgi recycling pathway requires the cytoplasmic domain acting together with the lumenal domain of ERGIC-53. Dissection of the cytoplasmic domain revealed a COOH-terminal di-lysine ER-retrieval signal, KKFF, and an RSQQE targeting determinant adjacent to the transmembrane domain. Surprisingly, the two COOH-terminal phenylalanines influence the targeting. They reduce the ER-retrieval capacity of the di-lysine signal and modulate the RSQQE determinant.
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Department of Pharmacology, University of Basel, Switzerland.
Schindler
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Hauri
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D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line / Q000378:metabolism; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism
D000939:Epitopes; C084004:LMAN1 protein, human; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D010649:Phenylalanine