http://www.jbc.org/content/272/12/7707.full.pdf

Calmodulin Regulates Endosome Fusion

Rab5, an early acting endosomal GTPase, supports in vitro endosome fusion without GTP hydrolysis.

Both Shiga holotoxin and the isolated B subunit, navigate a retrograde pathway from the plasma membrane to the endoplasmic reticulum (ER) of mammalian cells to deliver catalytic A subunits into the cytosol. This route passes through early/recycling endosomes and then through the Golgi. Although passage through the endosomes takes only 30 min, passage through the Golgi is much slower, taking hours. This suggests that Golgi passage is a key step in retrograde traffic. However, there is no empirical data demonstrating that Golgi passage is required for the toxins to enter the ER. In fact, an alternate pathway bypassing the Golgi is utilized by SV40 virus. Here we find that blocking Shiga toxin B access to the entire Golgi with AlF4− treatment, temperature block or subcellular surgery prevented Shiga toxin B from reaching the ER. This suggests that there is no direct endosome to ER route available for retrograde traffic. Curiously, when Shiga toxin B was trapped in endosomes, it entered the cytosol directly from the endosomal compartment. Our results suggest that trafficking through the Golgi apparatus is required for Shiga toxin B to reach the ER and that diversion into the Golgi may prevent toxin escape from endosomes into the cytosol.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1742-4658.2009.06890.x

Passage through the Golgi is necessary for Shiga toxin B subunit to reach the endoplasmic reticulum.

Summary

The acrosome reaction is a specialized exocytotic process. In the mouse there is compelling evidence that receptor-mediated activation of GTP-binding proteins by factors in the zona pellucida of oocytes is a central event in the acrosome reaction. Several reagents are able to affect GTP-binding proteins directly, bypassing the receptor–ligand step for activation. We have assessed the effect of several of these compounds on human spermatozoa, monitoring cell vitality and the acrosome reaction simultaneously using the triple-stain technique. GTPγS and aluminium fluoride complexes promote sperm activation very efficiently; amphiphilic peptides capable of activating Go and Gi, also elicit the acrosome reaction. The results indicate that activation of heterotrimeric GTP-binding proteins is sufficient to trigger acrosome exocytosis in human spermatozoa.

Reagents that activate GTP-binding proteins trigger the acrosome reaction in human spermatozoa.

The subcellular localization of the heterotrimeric G-proteins in hepatocytes in situ was compared to that in hepatocytes in primary culture. The ability of various ligands to activate adenylyl cyclase (AC) in membrane preparations was also investigated. In hepatocytes in situ the G proteins were mainly localized at the plasma membrane while in hepatocytes in culture they were predominantly cytoplasmic. The localization of the G-proteins in hepatocytes in situ correlates with their role in signal transduction. In homogenates prepared from the cultured cells, ligands which stimulate AC via Gs alpha were without effect, which was consistent with the localization of Gs alpha in the cytoplasmic and nuclear compartments. The "relocalization" of the G proteins to the cytoplasm when cells are cultured suggests that transmembrane signalling may be regulated by cell differentiation and cell-cell and cell-extracellular matrix interactions.

Comparison of the subcellular distribution of G-proteins in hepatocytes in situ and in primary cultures.

SummaryIt is now well established that GTP-binding proteins are important regulators of vesicular transport. Recent work has shown that multiple GTPases (both monomeric and heterotrimeric) are required for trafficking. In the present study we have used aluminum fluoride (AIF), a reagent that activates trimeric G proteins, as a tool to study the involvement of this family of GTPases in the regulation of endocytosis in intact cells. Our results indicate that AIF Inhibits fusion of early endosomes with an intracellular proteolytic compartment. Using the mixing of sequentially internalized ligands as a measure of endocytosis, we found that AIF inhibited endocytic transport as assessed by both biochemical and morphological methods. Taken together these results suggest that AIF affects membrane fusion, a common step in vesicular transport. To further examine the effects of AIF we tested this compound in a cell-free assay that reconstitutes fusion among endosomes. AIF affected endosomal fusion in a different way...

Inhibition of endocytic transport by aluminum fluoride implicates GTPases as regulators of endocytosis

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Inhibition of endocytic transport by aluminum fluoride implicates GTPases as regulators of endocytosis