Hsp47 and Cyclophilin B Traverse the Endoplasmic Reticulum with Procollagen into Pre-Golgi Intermediate Vesicles

We have previously demonstrated that endoplasmic reticulum (ER)-resident molecular chaperones interact with apolipoprotein B-100 (apoB) during its maturation. The initial stages of apoB folding occur while it is bound to the ER membrane, where it becomes partially lipidated to form a primordial intermediate. We determined whether this intermediate is dependent on the assistance of molecular chaperones for its subsequent folding steps. To that end, microsomes were prepared from HepG2 cells and luminal contents were subjected to KBr density gradient centrifugation. Immunoprecipitation of apoB followed by Western blotting showed that the luminal pool floated at a density of 1.12 g/ml and, like the membrane-bound pool, was associated with GRP94, ERp72, BiP, calreticulin, and cyclophilin B. Except for calreticulin, chaperone/apoB ratio in the lumen was severalfold higher than that in the membrane, suggesting a role for these chaperones both in facilitating the release of the primordial intermediate into the ER lumen and in providing stability. Subcellular fractionation on sucrose gradients showed that apoB in the Golgi was associated with the same array of chaperones as the pool of apoB recovered from heavy microsomes containing the ER, except that chaperone/apoB ratio was lower. KBr density gradient fractionation showed that the major pool of luminal apoB in the Golgi was recovered from 1.02 < d < 1.08 g/ml, whereas apoB in ER was recovered primarily from 1.08 < d < 1.2 g/ml. Both fractions were associated with the same spectrum of chaperones. Together with the finding that GRP94 was found associated with sialylated apoB, we conclude that correct folding of apoB is dependent on the assistance of molecular chaperone, which play multiple roles in its maturation throughout the secretory pathway including distal compartments such as the trans-Golgi network.The correct folding of apolipoprotein B (apoB) 1 into its mature, secretion-competent form is a complex process that leads to the formation and secretion of triacylglycerol (TAG)-rich lipoproteins, chylomicrons, and the atherogenic lipoproteins, very low density lipoproteins (VLDL) (1, 2). A two-step model for the formation of VLDL was originally proposed on the basis of electron microscopic studies in rat liver (3). This model was further supported by latter studies in rat liver (4), in rat hepatoma cells (McA-RH7777; Refs. 5-8), and in transgenic mice lacking the gene for apoB in the intestine (9). According to this model, the first step involves partial lipidation of apoB to form a primordial intermediate with HDL/LDL-like density. In the second step this intermediate fuses with a large apoB-free lipid droplet composed primarily of TAG to form nascent VLDL or chylomicrons. In HepG2 cells, however, these distinct steps were not clearly demonstrated. Nonetheless, the initial steps of lipidation are thought to be similar to other systems in that they are mediated by microsomal triglyceride transfer protein (MTP) (reviewed in Refs. 10 -12) and occur co-translationally,...

Section: Fig 5 Molecular Chaperones Co-immunoprecipitate With Apob supporting

confidence: 54%

Nascent Lipidated Apolipoprotein B Is Transported to the Golgi as an Incompletely Folded Intermediate as Probed by Its Association with Network of Endoplasmic Reticulum Molecular Chaperones, GRP94, ERp72, BiP, Calreticulin, and Cyclophilin B

Zhang

Herscovitz

2003

“…We do not know to what extent the substantial amount of these chaperones is due to a BFA effect or whether the ERGIC of nontreated cells also contains high levels of these chaperones in HepG2 cells. However, it is noticeable that the chaperone CBP1/HSP47 is known to be associated with procollagen in the ERGIC from where it is retrieved to the ER via its RDEL motif (64,80).…”

Section: Discussionmentioning

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

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.

“…This functional hypothesis is supported by a recent demonstration that Hsp47 binds specifically to stretches of non-hydroxylated Pro-Gly-X repeats (20), regions of low structural stability within the full-length collagen triple helices. After binding there is clear evidence that triple helical procollagen-Hsp47 complexes are eventually transported from the endoplasmic reticulum to cis-Golgi (21), where Hsp47 is dissociated and recycled back to the endoplasmic reticulum (18).…”

mentioning

confidence: 99%

The Molecular Interactions of Heat Shock Protein 47 (Hsp47) and Their Implications for Collagen Biosynthesis

Dafforn

Della

Miller

2001

108

Heat shock protein 47 (Hsp47) is a procollagen/collagen-specific molecular chaperone protein derived from the serpin family of proteins and essential for the early stages of collagen biosynthesis. In this paper, the results of an extensive biophysical analysis of mature recombinant mouse Hsp47 show the existence of both a structurally mesostable monomer with a 5-strand A-sheet and/or a hyperstable trimer; both states have biological activity. It is also demonstrated that Hsp47 is able to bind to a monomeric and partially folded conformation collagen mimic peptide (PPG) 10