An increasing number of plasma membrane proteins have been shown to be attached to the membrane via a glycosylphosphatidylinositol (GPI) moiety. All eukaryotes share a highly conserved GPI-core structure EthN-P-Man3-GlcN-PI, where EthN is ethanolamine. We have identified a protein encoded by the yeast open reading frame YGL142C that shares 33% identity with the human Pig-B protein. Deletion of this essential gene leads to a block in GPI anchor biosynthesis. We therefore named the gene GPI10. Gpi10p and Pig-B are functional homologues and the lethal deletion of GPI10 can be rescued by expression of the PIG-B cDNA. As found for PIG-B mutant cells, gpi10 deletant cells cannot attach the third mannose in an alpha-1,2 linkage to the GPI core-structure intermediate. Overexpression of GPI10 gives partial resistance to the GPI-synthesis inhibitor YW3548, suggesting that this gene product may affect the target of the inhibitor.

Epithelial-to-Mesenchymal Transition (EMT) is a key process contributing to the aggressiveness of cancer cells. EMT is triggered by activation of different transcription factors collectively known as EMT-TFs. Different cellular cues and cell signalling networks activate EMT at transcriptional and posttranscriptional level in different biological and pathological situations. Among them, overexpression of LOXL2 (lysyl oxidase-like 2) induces EMT independent of its catalytic activity. Remarkably, perinuclear/cytoplasmic accumulation of LOXL2 is a poor prognosis marker of squamous cell carcinomas and is associated to basal breast cancer metastasis by mechanisms no yet fully understood. Here, we report that overexpression of LOXL2 promotes its accumulation in the Endoplasmic Reticulum where it interacts with HSPA5 leading to activation of the IRE1-XBP1 signalling pathway of the ER-stress response. LOXL2-dependent IRE1-XBP1 activation induces the expression of several EMT-TFs: SNAI1, SNAI2, ZEB2 and TCF3 that are direct transcriptional targets of XBP1. Remarkably, inhibition of IRE1 blocks LOXL2-dependent upregulation of EMT-TFs thus hindering EMT induction.

The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI).Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen. Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively. We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole. We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein. Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions. Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles. Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13-0.27-m round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082⌬C32p, or lacking Apg12p. In contrast to the autophagy mutants involved in API transport, a ⌬yol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway. The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers. Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19.In the yeast Saccharomyces cerevisiae the vacuolar hydrolase leucine aminopeptidase I (API) 1 is synthesized in the cytoplasm as a precursor (pAPI) (1) and delivered to the vacuole by one of two alternative routes that operate under distinct physiological conditions: the cytoplasm to vacuole targeting (Cvt), in nutrient-rich conditions, and the autophagy (Apg) pathway, under starvation conditions (2). The Cvt pathway is constitutive and biosynthetic, while autophagy is nonselective and degradative and is induced to survive periods of nutrient limitation (3). However, the two pathways share many molecular components and both involve sequestration by double-membrane saccular structures of unknown origin that capture the load, close into vesicles, and then fuse with the vacuole (4). A major difference between these pathways appears to be the size and content of the transport vesicles. The Cvt vesicles exclude cytoplasm and are smaller than autophagosomes that engulf bulk cytoplasm and even organelles (2). Strikingly, despite all these differences, targeting of API to the vacuole is specific and saturable, both in vegetative growth conditions and under nitrogen deprivation (3), although the molecular detail...