The mammalian KDEL receptor is an integral membrane protein with seven hydrophobic regions. Fusion proteins comprising a 37-kDa N-glycosylation reporter fused downstream of amino-terminal fragments of the KDEL receptor with varying numbers of hydrophobic regions were synthesized in an in vitro translation system containing canine pancreatic microsomes. The luminal or cytosolic orientation of the reporter, and hence of the hydrophilic region to which it is fused, was inferred from the presence or absence of glycosylation, which occurs only in the lumen of the microsomes. The cytosolic orientation of the N and C termini was also confirmed immunocytochemically. Our results suggest that the KDEL receptor is inserted into the membrane with only six transmembrane domains and that both the amino and carboxy termini are located in the cytoplasm.Proteins resident in the lumen of the endoplasmic reticulum (ER) continually leave the compartment with the bulk flow of vesicular traffic to the Golgi complex (16). From the Golgi apparatus, they are specifically recovered by the retrograde pathway on the basis of their carboxy-terminal tetrapeptide sequence KDEL (or a closely related sequence), which is both necessary and sufficient for ER localization (17,18). Recently, it was demonstrated that the KDEL (HDEL) sequence is also sufficient to confer ER localization on membrane proteins with their C terminus in the lumen (6,23,24). The receptor for this signal was initially identified in Saccharomyces cerevisiae as a 26-kDa membrane protein encoded by the ERD2 gene (21), and closely related mammalian homologs have been identified (10,25). This receptor, which determines the specificity and capacity of the retention system (12, 21), has recently been shown to bind the KDEL ligand in vitro (26).Although the exact mechanism of the retrieval process is not known, it is believed that soluble ER proteins bind the receptor in the cis-Golgi. The ligand-occupied receptor then enters the retrograde pathway which delivers the complex to the ER (11). Here, the soluble proteins are released, possibly through a pH-dependent change in the binding activity of the receptor (26). The regenerated receptor then returns to the Golgi complex by bulk vesicular transport to reenter the cycle. We have recently demonstrated that the receptor is indeed localized to the cis-Golgi and can recycle between the Golgi complex and the ER (25).While much effort has been directed at elucidating the biochemical and molecular dynamics of the KDEL receptor, the transmembrane orientation of the protein has remained unaddressed. This knowledge is essential for detailed studies on the structure-function relationship of the receptor and could provide insights on the regions that might potentially interact with cytosolic factors involved in its intracellular migrations. We have therefore investigated the transmembrane topology of the KDEL receptor. The amino acid sequence deduced from its cDNA sequence exhibits seven hydrophobic regions (10, 25) (Fig. la) which have all be...

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Rab23, a novel member of the Rab family of small GTPases, has recently been identified in mesangial cells (MCs). Although Rab23 levels in MCs are associated with glomerular nephropathies, the exact physiological and pathological roles of Rab23 in MCs are unknown. In the present study, its roles in MCs were explored by performing proteomics and systems biology analyses in MCs after knockdown or overexpression of Rab23. Knockdown of Rab23 was achieved by transfecting MCs with a plasmid expressing short hairpin RNA against Rab23, while overexpression of Rab23 was accomplished by transfection with the wild-type, dominant negative, and constitutively active Rab23 gene constructs. The effects of different levels of Rab23 activity on proteome of various biological pathways were investigated. Gel-based proteomic approaches and systems biology tools, respectively, were used to identify the Rab23-regulated proteins and the functional pathways. Proteomic analysis revealed the potential roles for Rab23 in multiple processes, including G-protein signal transduction, transcription modulation, RNA stabilization, protein synthesis and degradation, cytoskeleton reorganization, anti-oxidation and detoxification, circadian rhythm regulation and phagocytosis. Bioinformatics analyses showed that Rab23 impacts on multiple biological networks in MCs. These data may shed light on the roles of Rab23 in mesangiopathy or MC damage.

We have raised a monoclonal antibody (mAb) (HFD9) that detects a 28 kDa protein (p28) enriched in the Golgi membrane. p28 was localized to the perinuclear Golgi region in all cell lines thus far examined. Its Golgi localization was confirmed by its colocalization with Golgi markers using indirect immunofluorescence microscopy. Immunogold labelling demonstrates that the majority of p28 was localized on the cis-Golgi and its associated structures. Two independent experiments demonstrate that the p28 epitope recognized by mAb HFD9 is exposed to the cytosol. Extraction of Golgi membranes with a variety of reagents revealed that p28 behaves like an integral membrane protein. mAb HFD9 thus defines a novel 28 kDa integral membrane protein on the cis-Golgi. To our knowledge, p28 represents the first integral membrane protein of the Golgi system identified via the antibody approach whose epitope is cytoplasmically-oriented and highly-conserved. Monoclonal antibody HFD9 will thus provide a useful tool for further studies on the cis side of the Golgi, which is not well characterised due to the lack of good markers.