Following entry into non-phagocytic HeLa cells, the facultative pathogen Salmonella typhimurium survives and replicates within a membrane-bound vacuole. Preceding the initiation of intracellular ...replication there is a lag phase, during which the bacteria modulate their environment. This phase is characterized by the rapid recycling of early endosomal proteins present on the nascent vacuole followed by the acquisition of a subset of lysosomal proteins. To gain a better understanding of the mechanism of intracellular survival, we have followed the biogenesis of the S.typhimurium-containing vacuole (SCV) in HeLa cells expressing different mutant forms of the small GTPase rab7. We demonstrate that the SCV recruits pre-existing lysosomal glycoproteins (Lgps) in a rab7-dependent manner, without directly interacting with lysosomes. We also show the transient accumulation, in the vicinity of the SCV, of novel rab7- and Lgp-containing vesicles containing very low amounts of cathepsin D. The size of these vesicles is dependent on rab7 activity, suggesting a role for rab7 in their homotypic fusion. Taken together, these results indicate that rab7 regulates SCV biogenesis during the phase characterized by the rapid acquisition of lysosomal proteins. We propose that SCV maturation involves its interaction with rab7/Lgp-containing vesicles which are possible intermediate cargo components of the late endocytic pathway.
EEA1, an Early Endosome-Associated Protein Mu, Fi-Tjen; Callaghan, Judy M.; Steele-Mortimer, Olivia ...
The Journal of biological chemistry,
06/1995, Letnik:
270, Številka:
22
Journal Article
Recenzirano
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Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have ...cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly α-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine “finger” motifs. The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.
Membrane transport is known to be regulated by protein phosphorylation and by small GTPases of the rab family. Using specific antibodies, we have identified a 55 kDa phosphorylated protein which ...co-immunoprecipitated with the cytosolic forms of rab5 and other rab proteins. We demonstrate, on the basis of its mobility in two-dimensional electrophoresis gels and its immunological properties, that this protein is rab GDI (p55/GDI). We also found that, a minor fraction of p55/GDI is membrane associated, but, whilst also complexed with rab proteins, it is not phosphorylated. On the basis of these data we suggest that the cycling of rab proteins between membranes and cytosol is regulated by phosphorylation of p55/GDI.
Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport. We have previously reported that rab5 regulates endosome‐endosome recognition and/or ...fusion in vitro. Here, we show that this process depends on the rab5 N‐terminal domain. Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide. Two‐dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions. Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin‐treatment of the cytosol. Trypsin‐treated cytosol prepared from mock‐transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments. Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N‐terminus. A synthetic peptide of rab5 N‐terminal domain inhibited endosome fusion in our cell‐free assay. A version of the same peptide truncated at the N‐terminus or a peptide of rab3 N‐terminal domain were without effects. Altogether, these observations suggest that the N‐terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery.
The rab5 protein is a member of the rab family of ras-related GTPases, which are known to be involved in the regulation of membrane transport in eukaryotic cells. Rab5 is localised to early ...endosomes, clathrin coated vesicles and the plasma membrane and has been shown to regulate an early step in the endocytic pathway both in vivo and in vitro. Although the exact function of the rab proteins is still unknown, their activity is believed to be dependent on their ability to cycle between GTP- and GDP-bound forms. A cell-free assay has been used to characterise the role of rab5 in early endosome fusion. All three isoforms, rab5a, rab5b and rab5c, showed the same in vitro activity. The nucleotide requirement of rab5a was investigated using mutant proteins. The results of these experiments indicate that GTP-binding is required for rab5 activity, and that hydrolysis is required for inactivation and recycling of the protein. Data was also obtained indicating that the N-terminal domain of rab5 is required for its function. Two phosphoproteins were detected which specifically co-immunoprecipitate with rab5. One of these was identified as the regulatory protein Rab-GDI. Although a small fraction of GDI was found on membranes, phosphorylated GDI was detected only in cytosol fractions. It is proposed that phosphorylation/dephosphorylation of GDI can regulate the specificity and directionality of the rab protein cycle. Finally, the ability of the REP-1 protein to deliver rab proteins to membranes was investigated. Purified REP-1/rab5 complex significantly stimulated endosome fusion, confirming the hypothesis that REP-1 is an escort protein. This system should now enable us to study in detail the requirements for GDI and REP proteins in rab protein regulation.
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