Effect of ATP depletion and DTT on the transport of membrane proteins from the endoplasmic reticulum and the intermediate compartment to the Golgi complex

Newly synthesized membrane proteins are exported from the endoplasmic reticulum to the Golgi complex through an intermediate compartment. Incubation st low temperature (15 degrees C) arrests the proteins in the intermediate compartment and prevents the entry into the Golgi complex. We have studied, in living cells, the effect of dithiothreitol (DTT) and ATP depletion on the transport to the Golgi complex of proteins accumulated either in the endoplasmic reticulum or in the intermediate compartment after a temperature block. The morphological results obtained with vesicular stomatitis virus ts-O45 G glycoprotein and the biochemical analysis performed with human CD8 protein, an O-glycosylated protein, showed that: i) ATP depletion blocks the export to the Golgi complex of proteins located either in the endoplasmic reticulum or in the intermediate compartment and ii) DTT interferes with the folding and export of proteins located in the endoplasmic reticulum, but it does not prevent the transfer from the intermediate compartment to the Golgi complex

Melanosome transfer promoted by keratinocyte growth factor in light and dark skin-derived keratinocytes.

The transfer of melanin from melanocytes to keratinocytes is upregulated by UV radiation and modulated by autocrine and paracrine factors. Among them, the keratinocyte growth factor (KGF/FGF7) promotes melanosome transfer acting on the recipient keratinocytes through stimulation of the phagocytic process. To search for possible differences in the melanosome uptake of keratinocytes from different skin color, we analyzed the uptake kinetics and distribution pattern of fluorescent latex beads in primary cultures of light and dark skin-derived keratinocytes stimulated with KGF and we compared the direct effect of KGF on the melanosome transfer in co-cultures of human primary melanocytes with light and dark keratinocytes. KGFpromoted melanosome transfer was more significant in light keratinocytes compared to dark, due to an increased expression of KGF receptor in light skin keratinocytes. Colocalization studies performed by confocal microscopy using FITC-dextran as a phagocytic marker and fluorescent beads as well as inhibition of particle uptake by cytochalasin D, revealed that beads internalization induced by KGF occurs via actin-dependent phagocytosis. 3D image reconstruction by fluorescence microscopy and ultrastructural analysis through transmission electron microscopy showed differences in the distribution pattern of the beads in light and dark keratinocytes, consistent with the different melanosome distribution in human skin

Shc proteins are localized on endoplasmic reticulum membranes and are redistributed following tyrosine kinase receptor activation.

The intracellular localization of She proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated,vith the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of She and erbB-2 kinase showed that receptor autophosphorylation, but not She phosphorylation, is required for redistribution of She proteins. The rough endoplasmic reticulum localization of She proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein

KDEL and KKXX Retrieval Signals Appended to the Same Reporter Protein Determine Different Trafficking between Endoplasmic Reticulum, Intermediate Compartment, and Golgi Complex

Many endoplasmic reticulum (ER) proteins maintain their residence by dynamic retrieval from downstream compartments of the secretory pathway. In previous work we compared the retrieval process mediated by the two signals, KKMP and KDEL, by appending them to the same neutral reporter protein, CD8, and found that the two signals determine a different steady-state localization of the reporter. CD8-K (the KDEL-bearing form) was restricted mainly to the ER, whereas CD8-E19 (the KKMP-bearing form) was distributed also to the intermediate compartment and Golgi complex. To investigate whether this different steady-state distribution reflects a difference in exit rates from the ER and/or in retrieval, we have now followed the first steps of export of the two constructs from the ER and their trafficking between ER and Golgi complex. Contrary to expectation, we find that CD8-K is efficiently recruited into transport vesicles, whereas CD8-E19 is not. Thus, the more restricted ER localization of CD8-K must be explained by a more efficient retrieval to the ER. Moreover, because most of ER resident CD8-K is not O-glycosylated but almost all CD8-E19 is, the results suggest that CD8-K is retrieved from the intermediate compartment, before reaching the Golgi, where O-glycosylation begins. These results illustrate how different retrieval signals determine different trafficking patterns and pose novel questions on the underlying molecular mechanisms

Adesione alle linee guida per il trattamento dei carcinomi della mammella a stadio limitato in relazione alla spesa sanitaria nazionale totale.

Adesione alle linee guida per il trattamento dei carcinomi della mammella a stadio limitato in relazione alla spesa sanitaria nazionale total

Indicators od compliance with guidelines in the EUROCARE-3 High Resolution study on breast cancer.

Indicators od compliance with guidelines on breast cance