We have identified CALNUC, an EF-hand, Ca2+-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gαi3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gαi3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca2+-binding protein detected by 45Ca2+-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.

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Autoimmune diseases are among the most prevalent of afflictions, yet the genetic factors responsible are largely undefined. Protein glycosylation in the Golgi apparatus produces structural variation at the cell surface and contributes to immune self-recognition. Altered protein glycosylation and antibodies that recognize endogenous glycans have been associated with various autoimmune syndromes, with the possibility that such abnormalities may reflect genetic defects in glycan formation. We show that mutation of a single gene, encoding ␣-mannosidase II, which regulates the hybrid to complex branching pattern of extracellular asparagine (N)-linked oligosaccharide chains (N-glycans), results in a systemic autoimmune disease similar to human systemic lupus erythematosus. ␣-Mannosidase IIdeficient autoimmune disease is due to an incomplete overlap of two conjoined pathways in complex-type N-glycan production. Lymphocyte development, abundance, and activation parameters are normal; however, serum immunoglobulins are increased and kidney function progressively falters as a disorder consistent with lupus nephritis develops. Autoantibody reactivity and circulating immune complexes are induced, and anti-nuclear antibodies exhibit reactivity toward histone, Sm antigen, and DNA. These findings reveal a genetic cause of autoimmune disease provoked by a defect in the pathway of protein N-glycosylation.autoimmunity ͉ genetics ͉ lupus ͉ glomerulonephritis

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Monitoring Editor: Suzanne R. Pfeffer RGS-GAIP (G␣-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate G␣ i /G␣ q -linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein G␣ i3 , which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular carrier fractions; it was not found in either Golgi-or PM-enriched fractions. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion. INTRODUCTIONClassical G protein-mediated signaling pathways are three-component systems consisting of serpentine (seven-transmembrane domain) plasma membrane (PM) 1 receptors, heterotrimeric G proteins composed of ␣, ␤, and ␥ subunits, and an effector, usually an enzyme or an ion channel (Gilman, 1987;Bourne et al., 1990;Neer, 1995;Hamm and Gilchrist, 1996). The newly discovered family of proteins known as RGS proteins (regulators of G protein signaling) constitute a fourth component of these systems (Dohlman and Thorner, 1997;Koelle, 1997;Neer, 1997;Berman and Gilman, 1998). RGS proteins serve as guanosine triphosphatase-activating proteins (GAPs) that accelerate the guanosine triphosphatase activity of G␣i/ G␣q subunits by stabilizing the G␣ subunit in its guanosine triphosphate (GTP)-to-guanosine diphosphate (GDP) transition state , returning them to their inactive GDP-bound form Hunt et al., 1996;Watson et al., 1996), and thereby terminating the G protein signal. The RGS protein family has been implicated in desensitization and negative regulation of heterotrimeric G proteinsignaling pathways in yeast, fungi, and nematodes (Dohlman et al., 1996;Koelle and Horvitz, 1996;Yu et al., 1996). In mammalian cells, RGS proteins have been implicated in the negative regulation of MAP kinase and phosphoinositide-phospholipase C activity and a loss of inhibition of adenylate cyclase activity by G␣i subunits Chatterjee et al., 1997;Huang et al., 1997;Yan et al., 1997). RGS proteins may also regulate cell death as suggested by the finding that A28-RGS14 is transcriptionally activated by the tumor ...