The chemotherapeutic agent paclitaxel inhibits autophagy through two distinct mechanisms that regulate apoptosis.

Anti-mitotic agents such as paclitaxel and docetaxel are widely used for the treatment of breast, ovarian and lung cancers. Although paclitaxel induces apoptosis, this drug also modulates autophagy. How autophagy affects paclitaxel activity, is unclear. We discovered that paclitaxel inhibited autophagy through two distinct mechanisms dependent on cell cycle stage. In mitotic cells, paclitaxel blocked activation of the class III phosphatidyl inositol 3 kinase, Vps34, a critical initiator of autophagosome formation. In non-mitotic paclitaxel-treated cells, autophagosomes were generated but their movement and maturation was inhibited. Chemically or genetically blocking autophagosome formation diminished paclitaxel-induced cell death suggesting that autophagosome accumulation sensitized cells to paclitaxel toxicity. In line with these observations, we identified that primary breast tumors that expressed diminished levels of autophagy-initiating genes were resistant to taxane therapy, identifying possible mechanisms and prognostic markers of clinical chemotherapeutic resistance.

Manipulation of oxidative protein folding and PDI redox state in mammalian cells.

In the endoplasmic reticulum (ER), disulfide bonds are simultaneously formed in nascent proteins and removed from incorrectly folded or assembled molecules. In this compartment, the redox state must be, therefore, precisely regulated. Here we show that both human Ero1-Lalpha and Ero1-Lbeta (hEROs) facilitate disulfide bond formation in immunoglobulin subunits by selectively oxidizing PDI. Disulfide bond formation is controlled by hEROs, which stand at a crucial point of an electron-flow starting from nascent secretory proteins and passing through PDI. The redox state of ERp57, another ER-resident oxidoreductase, is not affected by over-expression of Ero1-Lalpha, suggesting that parallel and specific pathways control oxidative protein folding in the ER. Mutants in the Ero1-Lalpha CXXCXXC motif act as dominant negatives by limiting immunoglobulin oxidation. PDI-dependent oxidative folding in living cells can thus be manipulated by using hERO variants.

Basolateral sorting of furin in MDCK cells requires a phenylalanine-isoleucine motif together with an acidic amino acid cluster.

Furin is a subtilisin-related endoprotease which processes a wide range of bioactive proteins. Furin is concentrated in the trans-Golgi network (TGN), where proteolytic activation of many precursor proteins takes place. A significant fraction of furin, however, cycles among the TGN, the plasma membrane, and endosomes, indicating that the accumulation in the TGN reflects a dynamic localization process. The cytosolic domain of furin is necessary and sufficient for TGN localization, and two signals are responsible for retrieval of furin to the TGN. A tyrosine-based (YKGL) motif mediates internalization of furin from the cell surface into endosomes. An acidic cluster that is part of two casein kinase II phosphorylation sites (SDSEEDE) is then responsible for retrieval of furin from endosomes to the TGN. In addition, the acidic EEDE sequence also mediates endocytic activity. Here, we analyzed the sorting of furin in polarized epithelial cells. We show that furin is delivered to the basolateral surface of MDCK cells, from where a significant fraction of the protein can return to the TGN. A phenylalanine-isoleucine motif together with the acidic EEDE cluster is required for basolateral sorting and constitutes a novel signal regulating intracellular traffic of furin.

The tyrosinase tail mediates sorting to the lysosomal compartment in MDCK cells via a di-leucine and a tyrosine-based signal.

Tyrosinase is a type I membrane protein found in melanosomes, which are lysosomal-like organelles and specific for pigment cells. A mutation of mouse tyrosinase, platinum (cp), leads to truncation of tyrosinase's cytosolic tail, and results in misrouting to the cell periphery. In this study, we expressed chimeras of wild-type and mutant cytosolic tails of mouse tyrosinase fused to rat lysosome-associated membrane protein-1 luminal and transmembrane domain to study sorting of tyrosinase in Madin-Darby canine kidney cells. The study shows that the mouse tyrosinase cytosolic tail is necessary and sufficient to mediate sorting of a heterologous type I membrane protein to compartments of the lysosomal lineage. Whereas deletions of 7 or 10 C-terminal amino acids of the tail still result in sorting to lysosomes, a deletion mutant corresponding to platinum (cp) tail fails to sort correctly and corroborates the in situ findings in cp homozygous mutant mice. Correct sorting of tyrosinase-lysosome-associated membrane protein-1 chimeras is mediated by the interplay of a di-leucine signal and a tyrosine motif of the Y-X-X-O type.

Trafficking of lysosomal membrane proteins in polarized kidney cells.

Segregation of lysosomal membrane proteins into the endosomal system occurs via clathrin coated vesicles, either from the trans-Golgi network or the cell surface. In both cases, cytosolic signals present in these proteins interact with clathrin adaptor proteins. In polarized kidney MDCK cells, transport of lysosomal membrane proteins via the plasma membrane occurs in a polarized fashion through the basolateral domain. Here, we discuss recent developments on lysosomal membrane protein trafficking, with a particular focus on the transport of these proteins in polarized MDCK cells.

IgM polymerization inhibits the Golgi-mediated processing of the mu-chain carboxy-terminal glycans.

Secreted glycoproteins generally contain oligosaccharides of the complex type. However, several molecules have been described in which individual glycans are processed differently from one another. Folding, assembly and oligomerization could affect the maturation of certain glycans by hindering them to the Golgi processing machinery. We have tested this possibility by analysing a panel of engineered murine mu chains secreted as mu2L2 monomers or as polymers, and having or not the carboxy-terminal glycan (Asn563). In secreted IgM polymers, Asn563 bears high-mannose oligosaccharides, typical of endoplasmic reticulum resident proteins, while complex sugars are found at the other four sites (Brenckle and Kornfeld, 1980 Arch. Biochem. Biophys. 243, 605-618). Polymeric and monomeric IgM contain mu chains whose glycans are processed differently. We show here that this is mainly due to the differential processing at the Asn563 glycan, which undergoes Golgi-mediated processing when IgM are secreted in the monomeric form. These results indicate that the oligomerization-dependent accessibility to the sugar modifying enzymes can be one of the key features that dictate the extent of oligosaccharide processing in multimeric glycoproteins. The presence of high mannose glycans at Asn563 implies that IgM polymerization takes place before encountering mannosidase II, likely in a pre-Golgi compartment.

trans-Golgi retention of a plasma membrane protein: mutations in the cytoplasmic domain of the asialoglycoprotein receptor subunit H1 result in trans-Golgi retention.

Unlike the wild-type asialoglycoprotein receptor subunit H1 which is transported to the cell surface, endocytosed and recycled, a mutant lacking residues 4-33 of the 40-amino acid cytoplasmic domain was found to be retained intracellularly upon expression in different cell lines. The mutant protein accumulated in the trans-Golgi, as judged from the acquisition of trans-Golgi-specific modifications of the protein and from the immunofluorescence staining pattern. It was localized to juxtanuclear, tubular structures that were also stained by antibodies against galactosyltransferase and gamma-adaptin. The results of further mutagenesis in the cytoplasmic domain indicated that the size rather than the specific sequence of the cytoplasmic domain determines whether H1 is retained in the trans-Golgi or transported to the cell surface. Truncation to less than 17 residues resulted in retention, and extension of a truncated tail by an unrelated sequence restored surface transport. The transmembrane segment of H1 was not sufficient for retention of a reporter molecule and it could be replaced by an artificial apolar sequence without affecting Golgi localization. The cytoplasmic domain thus appears to inhibit interaction(s) of the exoplasmic portion of H1 with trans-Golgi component(s) for example by steric hindrance or by changing the positioning of the protein in the membrane. This mechanism may also be functional in other proteins.