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Summary The initiation of cytotoxic immune responses by dendritic cells (DCs) requires the presentation of antigenic peptides derived from phagocytosed microbes and infected or dead cells to CD8 + T cells, a process called cross-presentation. Antigen cross-presentation by non-activated DCs, however, is not sufficient for the effective induction of immune responses. Additionally, DCs need to be activated through innate receptors, like Toll-like receptors (TLRs). During DC maturation, cross-presentation efficiency is first upregulated and then turned off. Here we show that during this transient phase of enhanced cross-presentation, phago-lysosome fusion was blocked by the topological re-organization of lysosomes into perinuclear clusters. LPS-induced lysosomal clustering, inhibition of phago-lysosome fusion and enhanced cross-presentation, all required expression of the GTPase Rab34. We conclude that TLR4 engagement induces a Rab34-dependent re-organization of lysosomal distribution that delays antigen degradation to transiently enhance cross-presentation, thereby optimizing the priming of CD8 + T cell responses against pathogens. [ABSTRACT FROM AUTHOR]

Hmunc13 is a cytosolic diacylglycerol (DAG)-binding protein, which is upregulated in renal cortical tubule and mesangial cells by hyperglycemia. In response to DAG activation, hmunc13 translocates to the Golgi. To investigate how this may relate to its function, we used a bacterial two-hybrid screen to look for hmunc13-interacting proteins. Full-length Rab34 was specifically isolated from a human kidney cDNA library. Co-expression of the two proteins confirmed Rab34 as a Golgi-associated protein, which was immunoprecipitated from cell lysates by hmunc13. Glutathione S-transferase fusion proteins of WT, Q111L (GTP bound), and T66N (GDP bound) mutants were created, and their GTP-binding activity verified by radioactive overlay assay. Binding of hmunc13 was observed with Q111L, barely detectable with T66N and enhanced with Rab34WT loaded with GTPγS compared with GDP loaded. Deletion of munc homolgy domain (MHD)-2, eliminated the hmunc13/Rab34 interaction. The Q111L mutant localized to the Golgi apparatus, but T66N was cytosolic. Localization of both mutants and Rab34WT was unchanged by DAG activation. The data suggest that DAG activation of hmunc13 causes it to be translocated to the Golgi, where it binds to GTP-bound Rab34 via MHD-2. Because Rab34 is known to regulate intracellular lysosome positioning, we propose that hmunc13 serves as an effector of Rab34, mediating lysosome–Golgi trafficking. [ABSTRACT FROM AUTHOR]