Hans-Peter Hauri

β-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo

Microinjection of antibodies against a synthetic peptide of a non-clathrin-coated vesicle-associated coat protein, beta-COP, blocks transport of a temperature-sensitive vesicular stomatitis virus glycoprotein (ts-O45-G) to the cell surface. Transport is inhibited upon release of the viral glycoprotein from temperature blocks at 39.5 degrees C (endoplasmic reticulum [ER]) and 15 degrees C (intermediate compartment), but not at 20 degrees C (trans-Golgi network). Ts-O45-G is arrested in tubular membrane structures containing p53 at the interface of the ER and the Golgi stack. This is consistent with inhibition of acquisition of endoglycosidase H resistance of ts-O45-G in injected cells. Secretion of endogenous proteins and maturation of cathepsin D are also inhibited. These data provide in vivo evidence that beta-COP has an important function in biosynthetic membrane traffic in mammalian cells.

The lectin ERGIC-53 is a cargo transport receptor for glycoproteins

Soluble secretory proteins are transported from the endoplasmic reticulum (ER) to the ER–Golgi intermediate compartment (ERGIC) in vesicles coated with COP-II coat proteins. The sorting of secretory cargo into these vesicles is thought to involve transmembrane cargo-receptor proteins. Here we show that a cathepsin-Z-related glycoprotein binds to the recycling, mannose-specific membrane lectin ERGIC-53. Binding occurs in the ER, is carbohydrate- and calcium-ion-dependent and is affected by untrimmed glucose residues. Binding does not, however, require oligomerization of ERGIC-53, although oligomerization is required for exit of ERGIC-53 from the ER. Dissociation of ERGIC-53 occurs in the ERGIC and is delayed if ERGIC-53 is mislocalized to the ER. These results strongly indicate that ERGIC-53 may function as a receptor facilitating ER-to-ERGIC transport of soluble glycoprotein cargo.

Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2)

We studied the postsynthetic sorting of endogenous plasma membrane proteins in a polarized epithelial cell line, Caco-2. Pulse-chase radiolabeling was combined with domain-specific cell surface assays to monitor the arrival of three apical and one basolateral protein at the apical and basolateral cell surface. Apical proteins were inserted simultaneously into both membrane domains. The fraction targeted to the basolateral domain was different for the three apical proteins and was subsequently sorted to the apical domain by transcytosis at different rates. In contrast, a basolateral protein was found in the basolateral membrane only. Thus, sorting of plasma membrane proteins occurred from two sites: the Golgi apparatus and the basolateral membrane. These data explain apparently conflicting results of earlier studies.

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin–Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N‐linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N‐linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N‐linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal‐mediated in polarized and non‐polarized cells.

Cellular COPII Proteins Are Involved in Production of the Vesicles That Form the Poliovirus Replication Complex

ABSTRACT Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway.

The endoplasmic reticulum—Golgi intermediate compartment

Abstract The recent identification of an endoplasmic reticulum-Golgi intermediate compartment has added to the complexity of the structural and functional organization of the early secretory pathway. Protein sorting along the endoplasmic reticulum-Golgi pathway depends on different signals and mechanisms, some of which guarantee recycling from various levels of the Golgi apparatus to biosynthetically earlier compartments.

Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells

An experimental model was designed to analyze the effect of fetal gut mesenchyme on the cytodifferentiation of crypt cells and of embryonic progenitor cells. The cells used were the rat intestinal crypt cell line, IEC-17, and primary cell cultures prepared form isolated 14-day-old fetal intestinal endoderm (EC). Both cultures prepared from isolated 14-day-old fetal rat intestinal endoderm (EC). Both types of cells were associated with 14-day-old fetal rat gut mesenchyme (Rm) and grafted under the kidney capsule of adult rats. Seventy percent of the Rm/EC and ten percent of the Rm/IEC recombinants, recovered after 9 days, exhibited well-vascularized structures in which the mesenchyme had induced morphogenesis of the cells into a villus epithelium. The four main intestinal epithelial cell types, absorptive, goblet, endocrine, and Paneth cells, were identified using electron microscopy. Biochemical determinations of enzyme activities associated with brush border membranes revealed that alkaline phosphatase, lactase, sucrase, and maltase were expressed in both types of associations. These results were confirmed by immunofluorescence staining using monoclonal antibodies to brush border enzymes. Both enzyme assays and immunocytochemistry showed that the amount of enzymes present in the brush border membrane of Rm/IEC grafts was in general lower than that of the Rm/EC recombinants. The results indicate that fetal rat gut mesenchyme enables morphogenesis and cytodifferentiation of both crypt and embryonic progenitor cells.

A novel direct interaction of endoplasmic reticulum with microtubules.

The positioning and dynamics of organelles in eukaryotic cells critically depend on membrane–cytoskeleton interactions. Motor proteins play an important role in the directed movement of organelle membranes along microtubules, but the basic mechanism by which membranes stably interact with the microtubule cytoskeleton is largely unknown. Here we report that p63, an integral membrane protein of the reticular subdomain of the rough endoplasmic reticulum (ER), binds microtubules in vivo and in vitro. Overexpression of p63 in cell culture led to a striking rearrangement of the ER and to concomitant bundling of microtubules along the altered ER. Mutational analysis of the cytoplasmic domain of p63 revealed two determinants responsible for these changes: an ER rearrangement determinant near the N‐terminus and a central microtubule‐binding region. The two determinants function independently of one another as indicated by deletion experiments. A peptide corresponding to the cytoplasmic tail of p63 promoted microtubule polymerization in vitro. p63 is the first identified integral membrane protein that can link a membrane organelle directly to microtubules. By doing so, it may contribute to the positioning of the ER along microtubules.

Role of Sec24 isoforms in selective export of membrane proteins from the endoplasmic reticulum

Sec24 of the COPII (coat protein complex II) vesicle coat mediates the selective export of membrane proteins from the endoplasmic reticulum (ER) in yeast. Human cells express four Sec24 isoforms, but their role is unknown. Here, we report the differential effects of Sec24 isoform‐specific silencing on the transport of the membrane reporter protein ERGIC‐53 (ER–Golgi intermediate compartment‐53) carrying the cytosolic ER export signals di‐phenylalanine, di‐tyrosine, di‐leucine, di‐isoleucine, di‐valine or terminal valine. Knockdown of single Sec24 isoforms showed dependence of di‐leucine‐mediated transport on Sec24A, but transport mediated by the other signals was not affected. By contrast, double knockdown of Sec24A with one of the other three Sec24 isoforms impaired all aromatic/hydrophobic signal‐dependent transport. Double knockdown of Sec24B/C or Sec24B/D preferentially affected di‐leucine‐mediated transport, whereas knockdown of Sec24C/D affected di‐isoleucine‐ and valine‐mediated transport. The isoform‐selective transport correlated with binding preferences of the signals for the corresponding isoforms in vitro. Thus, human Sec24 isoforms expand the repertoire of cargo for signal‐mediated ER export, but are in part functionally redundant.

Morphogenesis of the endoplasmic reticulum: beyond active membrane expansion.

The endoplasmic reticulum (ER) of higher eukaryotic cells is a dynamic network of interconnected membrane tubules that pervades almost the entire cytoplasm. On the basis of the morphological changes induced by the disruption of the cytoskeleton or molecular motor proteins, the commonly accepted model has emerged that microtubules and conventional kinesin (kinesin‐1) are essential determinants in establishing and maintaining the structure of the ER by active membrane expansion. Surprisingly, very similar ER phenotypes have now been observed when the cytoskeleton‐linking ER membrane protein of 63 kDa (CLIMP‐63) is mutated, revealing stable attachment of ER membranes to the microtubular cytoskeleton as a novel requirement for ER maintenance. Additional recent findings suggest that ER maintenance also requires ongoing homotypic membrane fusion, possibly controlled by the p97/p47/VICP135 protein complex. Work on other proteins proposed to regulate ER structure, including huntingtin, the EF‐hand Ca2+‐binding protein p22, the vesicle‐associated membrane protein‐associated protein B and kinectin isoforms further contribute to the new emerging concept that ER shape is not only determined by motor driven processes but by a variety of different mechanisms.