Felix Kappeler

The lectin ERGIC-53 is a cargo transport receptor for glycoproteins

Soluble secretory proteins are transported from the endoplasmic reticulum (ER) to the ER–Golgi intermediate compartment (ERGIC) in vesicles coated with COP-II coat proteins. The sorting of secretory cargo into these vesicles is thought to involve transmembrane cargo-receptor proteins. Here we show that a cathepsin-Z-related glycoprotein binds to the recycling, mannose-specific membrane lectin ERGIC-53. Binding occurs in the ER, is carbohydrate- and calcium-ion-dependent and is affected by untrimmed glucose residues. Binding does not, however, require oligomerization of ERGIC-53, although oligomerization is required for exit of ERGIC-53 from the ER. Dissociation of ERGIC-53 occurs in the ERGIC and is delayed if ERGIC-53 is mislocalized to the ER. These results strongly indicate that ERGIC-53 may function as a receptor facilitating ER-to-ERGIC transport of soluble glycoprotein cargo.

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin–Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N‐linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N‐linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N‐linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal‐mediated in polarized and non‐polarized cells.

A novel direct interaction of endoplasmic reticulum with microtubules.

The positioning and dynamics of organelles in eukaryotic cells critically depend on membrane–cytoskeleton interactions. Motor proteins play an important role in the directed movement of organelle membranes along microtubules, but the basic mechanism by which membranes stably interact with the microtubule cytoskeleton is largely unknown. Here we report that p63, an integral membrane protein of the reticular subdomain of the rough endoplasmic reticulum (ER), binds microtubules in vivo and in vitro. Overexpression of p63 in cell culture led to a striking rearrangement of the ER and to concomitant bundling of microtubules along the altered ER. Mutational analysis of the cytoplasmic domain of p63 revealed two determinants responsible for these changes: an ER rearrangement determinant near the N‐terminus and a central microtubule‐binding region. The two determinants function independently of one another as indicated by deletion experiments. A peptide corresponding to the cytoplasmic tail of p63 promoted microtubule polymerization in vitro. p63 is the first identified integral membrane protein that can link a membrane organelle directly to microtubules. By doing so, it may contribute to the positioning of the ER along microtubules.

Protein Targeting to Endoplasmic Reticulum by Dilysine Signals Involves Direct Retention in Addition to Retrieval

Dilysine signals confer localization of type I membrane proteins to the endoplasmic reticulum (ER). According to the prevailing model these signals target proteins to the ER by COP I-mediated retrieval from post-ER compartments, whereas the actual retention mechanism in the ER is unknown. We expressed chimeric membrane proteins with a C-terminal -Lys-Lys-Ala-Ala (KKAA) or -Lys-Lys-Phe-Phe (KKFF) dilysine signal in Lec-1 cells. Unlike KKFF constructs, which had access to post-ER compartments, the KKAA chimeras were localized to the ER by confocal microscopy and were neither processed by cis-Golgi-specific enzymes in vivonor included into ER-derived transport vesicles in an in vitro budding assay, suggesting that KKAA-bearing proteins are permanently retained in the ER. The ER localization was nonsaturable and exclusively mediated by the dilysine signal because mutating the lysines to alanines led to cell surface expression of the chimeras. Although the KKAA signal avidly binds COP I in vitro, the ER retention by this signal does not depend on intact COP I in vivo because it was not affected in an ε-COP-deficient cell line. We propose that dilysine ER targeting signals can mediate ER retention in addition to retrieval.

ER export of ERGIC-53 is controlled by cooperation of targeting determinants in all three of its domains

Selective export of proteins from the endoplasmic reticulum (ER) requires transport signals that have not been fully characterized. Here, we provide the first complete map of ER export determinants of a type I membrane protein, ERGIC-53, that cycles in the early secretory pathway. ER export requires a phenylalanine motif at the C-terminus, known to mediate coat protein II (COPII) interaction, that is assisted by a glutamine in the cytoplasmic domain. Disulfide bond-stabilized oligomerization is also required. Efficient hexamerization depends on the presence of a polar and two aromatic residues in the transmembrane domain (TMD). Oligomerization becomes independent on disulfide bonds when TMD hydrophobicity is increased. ER export is also influenced by TMD length, 21 amino acids being most efficient. When transferred to a signal-less construct, the established targeting motifs reconstitute full transport activity. The results suggest an ER-export mechanism in which transmembrane and luminal determinants mediate oligomerization required for efficient recruitment of ERGIC-53 into budding vesicles via the C-terminal COPII-binding phenylalanine motif.

A novel endocytosis signal related to the KKXX ER-retrieval signal

Membrane proteins often contain a sorting signal in their cytoplasmic tail that promotes their clustering into coated vesicles at a specific cellular site. ERGIC‐53 contains a cytoplasmic ER‐retrieval signal, KKFF. However, overexpressed ERGIC‐53 is transported to the cell surface and rapidly endocytosed. Here we report that ERGIC‐53 carries a previously undescribed endocytosis signal. Surprisingly, the signal was KKFF and like the ER‐retrieval signal required a C‐terminal position. In fact, the minimal consensus sequence determined by substitutional mutagenesis (K‐K/R‐F/Y‐F/Y) was related to the ER‐retrieval consensus (K‐K‐X‐X). Furthermore, we provide evidence that internalization of VIP36, a protein that cycles between plasma membrane and Golgi, is mediated by a signal at its C‐terminus that matches the internalization consensus sequence. The relatedness of the two signals suggests that coatomer‐mediated retrieval of proteins may be mechanistically more related to clathrin‐dependent sorting than previously anticipated.