The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein

113

Cycling proteins play important roles in the organization and function of the early secretory pathway by participating in membrane traffic and selective transport of cargo between the endoplasmic reticulum (ER), the intermediate compartment (ERGIC), and the Golgi. To identify new cycling proteins, we have developed a novel procedure for the purification of ERGIC membranes from HepG2 cells treated with brefeldin A, a drug known to accumulate cycling proteins in the ERGIC. Membranes enriched 110-fold over the homogenate for ERGIC-53 were obtained and analyzed by mass spectrometry. Major proteins corresponded to established and putative cargo receptors and components mediating protein maturation and membrane traffic. Among the uncharacterized proteins, a 32-kDa protein termed ERGIC-32 is a novel cycling membrane protein with sequence homology to Erv41p and Erv46p, two proteins enriched in COPII vesicles of yeast. ERGIC-32 localizes to the ERGIC and partially colocalizes with the human homologs of Erv41p and Erv46p, which mainly localize to the cis-Golgi. ERGIC-32 interacts with human Erv46 (hErv46) as revealed by covalent cross-linking and mistargeting experiments, and silencing of ERGIC-32 by small interfering RNAs increases the turnover of hErv46. We propose that ERGIC-32 functions as a modulator of the hErv41-hErv46 complex by stabilizing hErv46. Our novel approach for the isolation of the ERGIC from BFA-treated cells may ultimately lead to the identification of all proteins rapidly cycling early in the secretory pathway.

Section: Purification Of Ergic Membranes From Bfa-treated Cells-mentioning

confidence: 99%

Proteomics of Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC) Membranes from Brefeldin A-treated HepG2 Cells Identifies ERGIC-32, a New Cycling Protein That Interacts with Human Erv46

Breuza

Halbeisen

Jenö

et al. 2004

113

“…There is a growing consensus that also the Golgi apparatus can function as an agonist-releasable Ca 2ϩ store (6), the importance of which may, however, greatly differ among the different cell types and ranges from relatively unimportant in Drosophila melanogaster S2 cells (7) to very important in renal LLC-PK 1 cells (8). The storage capacity for Ca 2ϩ in the Golgi is greatly increased by specific proteins like Calnuc/nucleobindin (9,10) and members of the CREC family of Ca 2ϩ -binding proteins (reviewed in Ref. 11).…”

Section: )mentioning

confidence: 99%

The Golgi PMR1 P-type ATPase of Caenorhabditis elegans

Baelen

Vanoevelen

Missiaen

et al. 2001

In recent years, it has been well established that the Ca 2؉ concentration in the lumen of intracellular organelles is a key determinant of cell function. Despite the fact that essential functions of the Golgi apparatus depend on the Ca 2؉ and Mn 2؉ concentration in its lumen, little is known on the transport system responsible for ion accumulation. The Golgi ion pump PMR1 has been functionally studied only in yeast. In humans, mutations in the orthologous gene ATP2C1 cause HaileyHailey disease. We report here the identification of the PMR1 homologue in the model organism Caenorhabditis elegans and after ectopic expression the direct study of its ion transport in permeabilized COS-1 cells. The C. elegans genome is predicted to contain a single PMR1 orthologue on chromosome I. We found evidence for alternative splicing in the 5-untranslated region, but no indication for the generation of different protein isoforms. C. elegans PMR1 overexpressed in COS-1 cells transports Ca 2؉ and Mn 2؉ with high affinity into the Golgi apparatus in a thapsigargin-insensitive manner. Part of the accumulated Ca 2؉ can be released by inositol 1,4,5-trisphosphate, in agreement with the idea that the Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca 2؉ store.

“…3) are consistent with the notion that intra-Golgi transport depends upon Ca 2ϩ efflux from inner membrane stores. Several studies have indicated that the Golgi complex may function as a Ca 2ϩ storage organelle (8,(27)(28)(29). Thus it is feasible that Ca 2ϩ from the lumen of the Golgi directly participated in regulating intra-Golgi transport.…”

Section: Figmentioning

confidence: 99%

Regulation of Intra-Golgi Membrane Transport by Calcium

Porat

Elazar

2000

Calcium cations play a critical role in regulating vesicular transport between different intracellular membrane-bound compartments. The role of calcium in transport between the Golgi cisternae, however, remains unclear. Using a well characterized cell-free intra-Golgi transport assay, we now show that changes in free Ca 2؉ concentration in the physiological range regulate this transport process. The calcium-chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid blocked transport with an IC 50 of approximately 0.8 mM. The effect of 1,2-bis(2-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid was reversible by addition of fresh cytosol and was irreversible when performed in the presence of a Ca 2؉ ionophore that depletes calcium from lumenal stores. We demonstrate here that intra-Golgi transport is stimulated by low Ca 2؉ concentrations (20 -100 nM) but is inhibited by higher concentrations (above 100 nM). Further, we show that calmodulin antagonists specifically block intraGolgi transport, implying a role for calmodulin in mediating the effect of calcium. Our results suggest that Ca 2؉