Seng Hui Low

The mammalian homolog of yeast Sec13p is enriched in the intermediate compartment and is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus.

The role of COPII components in endoplasmic reticulum (ER)-Golgi transport, first identified in the yeast Saccharomyces cerevisiae, has yet to be fully characterized in higher eukaryotes. A human cDNA whose predicted amino acid sequence showed 70% similarity to the yeast Sec13p has previously been cloned. Antibodies raised against the human SEC13 protein (mSEC13) recognized a cellular protein of 35 kDa in both the soluble and membrane fractions. Like the yeast Sec13p, mSEC13 exist in the cytosol in both monomeric and higher-molecular-weight forms. Immunofluorescence microscopy localized mSEC13 to the characteristic spotty ER-Golgi intermediate compartment (ERGIC) in cells of all species examined, where it colocalized well with the KDEL receptor, an ERGIC marker, at 15 degrees C. Immunoelectron microscopy also localized mSEC13 to membrane structures close to the Golgi apparatus. mSEC13 is essential for ER-to-Golgi transport, since both the His6-tagged mSEC13 recombinant protein and the affinity-purified mSEC13 antibody inhibited the transport of restrictive temperature-arrested vesicular stomatitis virus G protein from the ER to the Golgi apparatus in a semi-intact cell assay. Moreover, cytosol immunodepleted of mSEC13 could no longer support ER-Golgi transport. Transport could be restored in a dose-dependent manner by a cytosol fraction enriched in the high-molecular-weight mSEC13 complex but not by a fraction enriched in either monomeric mSEC13 or recombinant mSEC13. As a putative component of the mammalian COPII complex, mSEC13 showed partially overlapping but mostly different properties in terms of localization, membrane recruitment, and dynamics compared to that of beta-COP, a component of the COPI complex.

Effects of NH4Cl and nocodazole on polarized fibronectin secretion vary amongst different epithelial cell types

The extracellular matrix protein fibronectin was found to be secreted by three polarized epithelial cell lines Madin-Darby canine kidney (MDCK), Caco-2 and LLC-PK1. About 54 and 46% of fibronectin was secreted from the apical and basolateral cell surfaces, respectively, in MDCK cells. In Caco-2 and LLC-PK1 cells, the majority (about 92-93%) of fibronectin secretion occurs from the basolateral cell surface, with the remaining 7-8% from the apical surface. In all three cell types, NH4Cl was found to inhibit basolateral secretion (resulting in enhanced apical secretion), while total fibronectin secretion was not significantly affected (although a delay in secretion was observed). Nocodazole reduced total fibronectin secretion to about 70% of control levels in MDCK and Caco-2 cells, with significant inhibition on secretion from both surfaces. In contrast, total fibronectin secretion was enhanced by nocodazole in LLC-PK1 cells. Furthermore, the majority of fibronectin secretion was redirected to the apical cell surface in LLC-PK1 cells. These observations demonstrate that the nature as well as the extent of the effects of NH4-Cl and nocodazole on polarized fibronectin secretion varies amongst different epithelial cell types.