Biomedical subjects

J Füllekrug

Publications and source records attributed to J Füllekrug.

8 recordsLinked to original sources

PubMed1999

Localization and recycling of gp27 (hp24gamma3): complex formation with other p24 family members.

We report here the characterization of gp27 (hp24gamma3), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secretory pathway. Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to the cis side of the Golgi apparatus. In addition, some gp27 was detected in COPI- and COPII-coated structures throughout the cytoplasm. This indicated cycling that was confirmed in three ways. First, 15 degrees C temperature treatment resulted in accumulation of gp27 in pre-Golgi structures colocalizing with anterograde cargo. Second, treatment with brefeldin A caused gp27 to relocate into peripheral structures positive for both KDEL receptor and COPII. Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticulum (ER) by blocking ER export. Together, this shows that gp27 cycles extensively in the early secretory pathway. Immunoprecipitation and coexpression studies further revealed that a significant fraction of gp27 existed in a hetero-oligomeric complex. Three members of the p24 family, GMP25 (hp24alpha2), p24 (hp24beta1), and p23 (hp24delta1), coprecipitated in what appeared to be stochiometric amounts. This heterocomplex was specific. Immunoprecipitation of p26 (hp24gamma4) failed to coprecipitate GMP25, p24, or p23. Also, very little p26 was found coprecipitating with gp27. A functional requirement for complex formation was suggested at the level of ER export. Transiently expressed gp27 failed to leave the ER unless other p24 family proteins were coexpressed. Comparison of attached oligosaccharides showed that gp27 and GMP25 recycled differentially. Only a very minor portion of GMP25 displayed complex oligosaccharides. In contrast, all of gp27 showed modifications by medial and trans enzymes at steady state. We conclude from these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibrium with individual p24 proteins to allow for differential recycling and distributions.

Amino Acid Sequence↗

PubMed1999

VIP36 localisation to the early secretory pathway.

VIP36, an integral membrane protein previously isolated from epithelial MDCK cells, is an intracellular lectin of the secretory pathway. Overexpressed VIP36 had been localised to the Golgi complex, plasma membrane and endocytic structures suggesting post-Golgi trafficking of this molecule (Fiedler et al., 1994). Here we provide evidence that endogenous VIP36 is localised to the Golgi apparatus and the early secretory pathway of MDCK and Vero cells and propose that retention is easily saturated. High resolution confocal microscopy shows partial overlap of VIP36 with Golgi marker proteins. Punctate cytoplasmic structures colocalise with coatomer and ERGIC-53, labeling ER-Golgi intermediate membrane structures. Cycling of VIP36 is suggested by colocalisation with anterograde cargo trapped in pre-Golgi structures and modification of its N-linked carbohydrate by glycosylation enzymes of medial Golgi cisternae. Furthermore, after brefeldin A treatment VIP36 is segregated from resident Golgi proteins and codistributes with ER-Golgi recycling proteins.

1-Deoxynojirimycin↗

PubMed1998

Protein sorting in the Golgi complex.

Even after one hundred years, the Golgi apparatus remains a major challenge in the field of Cell Biology. This is particularly true in terms of transport and of protein sorting. For example, the question how cargo proteins are transported through this organelle is still a matter of debate. Emphasis has been put on the role of anterograde and retrograde transport vesicles. These have been proposed to carry cargo from cisterna to cisterna and to recycle components needed for further rounds of transport. Alternatively, anterograde movement of cargo takes place in cisternal membranes rather than transport vesicles. These membranes assemble and mature in a cis to trans direction. In this case, retrograde transport vesicles need to recycle all components of the Golgi apparatus and this demands a highly dynamic and efficient sorting machinery. Here we will discuss possible mechanisms for protein sorting in the context of cisternal maturation and propose that a common mechanism is sufficient to explain both transport of cargo and sorting of resident proteins.

Carrier Proteins↗

PubMed1998

gp25L/emp24/p24 protein family members of the cis-Golgi network bind both COP I and II coatomer.

Abstract. Five mammalian members of the gp25L/ emp24/p24 family have been identified as major constituents of the cis-Golgi network of rat liver and HeLa cells. Two of these were also found in membranes of higher density (corresponding to the ER), and this correlated with their ability to bind COP I in vitro. This binding was mediated by a K(X)KXX-like retrieval motif present in the cytoplasmic domain of these two members. A second motif, double phenylalanine (FF), present in the cytoplasmic domain of all five members, was shown to participate in the binding of Sec23 (COP II). This motif is part of a larger one, similar to the F/YXXXXF/Y strong endocytosis and putative AP2 binding motif. In vivo mutational analysis confirmed the roles of both motifs so that when COP I binding was expected to be impaired, cell surface expression was observed, whereas mutation of the Sec23 binding motif resulted in a redistribution to the ER. Surprisingly, upon expression of mutated members, steady-state distribution of unmutated ones shifted as well, presumably as a consequence of their observed oligomeric properties.

Amino Acid Sequence↗

PubMed1997

Characterization of brefeldin A induced vesicular structures containing cycling proteins of the intermediate compartment/cis-Golgi network.

Residence of luminal ER proteins is mediated by a cyclic process which involves binding of escaped proteins to a KDEL receptor in a post-ER compartment and redistribution of the ligand-receptor complex back to the ER. We examined the relocation of the KDEL receptor after treatment with the fungal metabolite brefeldin A and compared this with the retrograde transport of the KDEL receptor observed after ligand or receptor overexpression. Incubation with brefeldin A led to the formation of vesicular structures containing the KDEL receptor and ERGIC-53, a marker for the ER-Golgi intermediate compartment. Immunoelectron microscopy revealed that these structures are composed of tubulo-vesicular clusters. The brefeldin A induced vesicular structures were morphologically and biochemically distinct from the ER-Golgi hybrid compartment as demonstrated by double immunofluorescence microscopy and subcellular fractionation. Overexpression of the receptor itself or a lysozyme-KDEL construct led to a shift of the KDEL receptor together with ERGIC-53, an intermediate compartment marker to the ER but not to structures resembling BFA induced vesicular structures. Moreover, overexpression of the receptor resulted in the partial redistribution of marker proteins of the medial Golgi and the trans-Golgi network to ER-like structures. We conclude that the effects of brefeldin A on the redistribution of the KDEL receptor do not reflect physiological events occurring during increased occupancy of the receptor with ligands.

Animals↗

PubMed1997

Human Rer1 is localized to the Golgi apparatus and complements the deletion of the homologous Rer1 protein of Saccharomyces cerevisiae.

Sec12p is a type II membrane glycoprotein in the endoplasmic reticulum (ER) of Saccharomyces cerevisiae which is essential for transport vesicle budding. It is the guanine nucleotide exchange factor for the small GTP-binding protein Sar1p which is a constituent of COP II ER to Golgi vesicles. We report the sequence and localization of the human homologue to yeast Rer1p, which has recently been identified genetically as an essential component for retention of Sec12p in the ER. Reverse polymerase chain reaction was used to obtain cDNAs from HeLa cells. They code for a protein of 196 amino acids, corresponding to a molecular mass of 23 kDa. The translated sequence is 44% identical and 65% similar to yeast Rer1 protein. The four putative transmembrane domains are predicted to form a W-topology with both N- and C-terminus facing the cytosol. The functional activity of myc-tagged human Rer1 was demonstrated by the complementation of the RER1 deletion in S. cerevisiae. Mislocalization of the Sec12-reporter protein was reduced similar to the results obtained with yeast Rer1p. Human Rer1 protein was expressed in HeLa cells and the subcellular distribution analyzed by double immunofluorescence and immunoelectron microscopy of thawed cryosections. The tagged protein was localized to the Golgi apparatus and peripheral elements of the ER-Golgi interface. High overexpression leads to relocation of human Rer1 to ER-like structures together with KDEL-receptor and affects the structural organization of the Golgi apparatus. Under conditions of brefeldin A treatment, human Rer1 distributes together with recycling Golgi proteins.

Adaptor Proteins, Vesicular Transport↗

PubMed1994

Retention and retrieval: both mechanisms cooperate to maintain calreticulin in the endoplasmic reticulum.

Many soluble resident proteins of the endoplasmic reticulum share a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. Current opinion favours a model in which these proteins can escape from the endoplasmic reticulum (ER) by bulk flow and are recognized and sorted in the Golgi apparatus by binding to a specific KDEL-receptor, which returns them to the ER. Through biochemical, morphological and mutational analysis we have studied the mechanisms that determine the localization of calreticulin, a soluble 60 kDa KDEL-protein of the ER. Immunogold labelling established the ER localization of calreticulin in transfected and nontransfected COS cells. Although the ER cisternae in transfected cells were enormously dilated and heavily labelled by gold particles we found no significant label in any other compartment. In vivo pulse chase experiments with [35S]methionine followed by biochemical fractionation of calreticulin overexpressing COS cells (50- to 100-fold) revealed that only a minor part of labelled calreticulin leaves the ER. Retrieval from the Golgi was confirmed by a partial redistribution of the endogenous KDEL-receptor as shown by double immunofluorescence. These data suggest a KDEL-independent retention of calreticulin in the ER. Further supporting evidence has come from morphological in vivo studies using calreticulin-transfected and vesicular stomatitis virus (ts045)-infected COS cells. Stimulation of vesicular transport from the ER by releasing the temperature-dependent transport block for the viral G-protein resulted in a small but significant appearance of calreticulin in a post-ER compartment. In contrast a calreticulin mutant, which lacked the Ca(2+)-binding domain but included the KDEL sequence, could escape from the ER to a much higher extent. Secretion of the nonmutated calreticulin was very low (1-2% of total calreticulin in 3 hours) compared to the mutated form (18% in 3 hours). Deletion of the KDEL sequence led to an increase in secretion to 29% over a 3 hour period, which is much less than expected for a secretory protein. Taken together these results strongly support the hypothesis of two independently operating retention/retrieval mechanisms for calreticulin: one providing for direct retention in the ER with a very high capacity and having Ca(2+)-dependent properties; the other a KDEL-based retrieval system for escaped calreticulin present in the Golgi apparatus.

Amino Acid Sequence↗

PubMed1994

CaBP1, a calcium binding protein of the thioredoxin family, is a resident KDEL protein of the ER and not of the intermediate compartment.

A cDNA encoding rat CaBP1 has been isolated and sequenced. The deduced polypeptide chain consists of 440 amino acids including two internal thioredoxin-like domains and a C-terminal KDEL retention/retrieval signal. Regarding the high degree of identity to the hamster protein P5, CaBP1 is considered to be the homologous rat protein. Previous work has suggested that CaBP1 is a resident luminal protein of the intermediate compartment (Schweizer, A., Peter, F., Nguyen Van, P., Söling, H.D. and Hauri, H.P. (1993) Eur. J. Cell Biol. 60, 366-370). Our conclusion that CaBP1 is a resident protein of the endoplasmic reticulum and not of the intermediate compartment is based on three different approaches: subcellular fractionation, indirect immunofluorescence and overexpression of CaBP1. Subcellular fractionation of Vero cells in a velocity controlled step gradient led to copurification of CaBP1-containing vesicles and several marker proteins for the ER including calreticulin and alpha-SSRP. The intermediate compartment, as defined by a monoclonal antibody against the marker protein p53 (ERGIC-53), could be separated from these ER markers. Double immunofluorescence analysed by laser scanning microscopy showed no significant colocalization between CaBP1 and p53, but between CaBP1 and calreticulin. In addition experiments, Vero cells were infected with VSV tsO45. At 15 degrees C the VSV-G protein accumulated in punctuate structures representing the intermediate compartment, while CaBP1 maintained its original reticular localization. Even after high-level overexpression in COS cells, CaBP1 was not detected in the intermediate compartment, but was efficiently retained in the ER as judged by light microscopy.

Amino Acid Sequence↗