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

Abstract

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.

The Cargo Receptors Surf4, Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC)-53, and p25 Are Required to Maintain the Architecture of ERGIC and Golgi

“…N-terminally tagged Surf4 localizes to the ER, whereas C-terminally tagged Surf4 localizes to the Golgi in transfected cells (Reeves and Fried, 1995). In contrast, endogenous Surf4 was identified by mass spectrometry in an ERGIC fraction isolated BFA-treated HepG2 cells (Breuza et al, 2004).…”

“…Five days after confluence, HepG2 cells were treated with 10 g/ml brefeldin A (BFA; Epicenter, Madison, WI) for 90 min, and ERGIC membranes were isolated by subcellular fractionation by using Nycodenz gradients (Breuza et al, 2004). Fractions of the Nycodenz gradient enriched in the ERGIC marker ERGIC-53 were pooled and diluted five times with phosphate-buffered saline (PBS).…”

“…Both nontransfected and HA-Surf4 -transfected cells were analyzed. ERGIC membranes were isolated by Nycodenz gradient centrifugation (Breuza et al, 2004), and the gradient fractions were analyzed for organelle markers by Western blotting. Surf4 largely codistributed with the ERGIC marker ERGIC-53 (Supplemental Figure S2).…”

“…HepG2 cells and HepG2 cells stably expressing HA-Surf4 (Breuza et al, 2004) were grown in minimal essential medium, supplemented with 10% fetal bovine serum. For metabolic labeling and immunoblotting cells were grown in six-well plates.…”

“…N-terminally tagged Surf4 localizes to the ER, whereas C-terminally tagged Surf4 localizes to the Golgi in transfected cells (Reeves and Fried, 1995). In contrast, endogenous Surf4 was identified by mass spectrometry in an ERGIC fraction isolated BFA-treated HepG2 cells (Breuza et al, 2004).To characterize endogenous Surf4, we prepared polyclonal antibodies to the N-terminal 60 amino acids of Surf4 fused to GST, and we purified them by affinity chromatography. Affinity-purified anti-Surf4 recognized a protein of ϳ22 kDa on Western blots in reasonable agreement with the Mr of Surf4 deduced from conceptual translation (Supplemental Figure S1A).…”