Florence Jollivet

Characterization of GAPCenA, a GTPase activating protein for Rab6, part of which associates with the centrosome.

The Rab6 GTPase regulates intracellular transport at the level of the Golgi apparatus, probably in a retrograde direction. Here, we report the identification and characterization of a novel human Rab6‐interacting protein named human GAPCenA (for ‘GAP and centrosome‐associated’). Primary sequence analysis indicates that GAPCenA displays similarities, within a central 200 amino acids domain, to both the yeast Rab GTPase activating proteins (GAPs) and to the spindle checkpoint proteins Saccharomyces cerevisiae Bub2p and Schizosaccharomyces pombe Cdc16p. We demonstrate that GAPCenA is indeed a GAP, specifically active in vitro on Rab6 and, to a lesser extent, on Rab4 and Rab2 proteins. Immunofluorescence and cell fractionation experiments showed that GAPCenA is mainly cytosolic but that a minor pool is associated with the centrosome. Moreover, GAPCenA was found to form complexes with cytosolic γ‐tubulin and to play a role in microtubule nucleation. Therefore, GAPCenA may be involved in the coordination of microtubule and Golgi dynamics during the cell cycle.

The human kinesin-like protein RB6K is under tight cell cycle control and is essential for cytokinesis

ABSTRACT Several members of the kinesin superfamily are known to play a prominent role in the motor-driven transport processes that occur in mitotic cells. Here we describe a new mitotic human kinesin-like protein, RB6K (Rabkinesin 6), distantly related to MKLP-1. Expression of RB6K is regulated during the cell cycle at both the mRNA and protein level and, similar to cyclin B, shows a maximum during M phase. Isolation of the RB6K promoter allowed identification of a CDE-CHR element and promoter activity was shown to be maximal during M phase. Immunofluorescence microscopy using antibodies raised against RB6K showed a weak signal in interphase Golgi but a 10-fold higher signal in prophase nuclei. During M phase, the newly synthesized RB6K does not colocalise with Rab6. In later stages of mitosis RB6K localized to the spindle midzone and appeared on the midbodies during cytokinesis. The functional significance of this localization during M phase was revealed by antibody microinjection studies which resulted exclusively in binucleate cells, showing a complete failure of cytokinesis. These results substantiate a crucial role for RB6K in late anaphase B and/or cytokinesis, clearly distinct from the role of MKLP-1.

Characterization of novel Rab6-interacting proteins involved in endosome-to-TGN transport.

Rab6 GTPase regulates intracellular transport at the level of the Golgi complex. Using the yeast two‐hybrid screen, we have isolated two clones that specifically interact with the three isoforms of Rab6 present in mammalian cells (Rab6A, A′ and B). The cDNAs encode two proteins of 976 and 1120 amino acids (calculated molecular mass of 112 and 128 kDa, respectively) that we named Rab6IP2A and Rab6IP2B (for Rab6 Interacting Protein 2). The two proteins likely correspond to spliced variants of the same gene. Rab6IP2s have no significant homology with other known proteins, including Rab effectors or partners. They are ubiquitously expressed, mostly cytosolic and found in high molecular mass complexes in brain cytosol. We show that Rab6IP2s can be recruited on Golgi membranes in a Rab6:GTP‐dependent manner. The overexpression of any form of Rab6IP2 has no detectable effect on the secretory pathway. In contrast, the retrograde transport of the Shiga toxin B subunit between the plasma membrane and the Golgi complex is partly inhibited in cells overexpressing the Rab6‐binding domain of Rab6IP2. Our data suggest that Rab6IP2s is involved in the pathway regulated by Rab6A′.

Synchronization of secretory protein traffic in populations of cells

To dissect secretory traffic, we developed the retention using selective hooks (RUSH) system. RUSH is a two-state assay based on the reversible interaction of a hook protein fused to core streptavidin and stably anchored in the donor compartment with a reporter protein of interest fused to streptavidin-binding peptide (SBP). Biotin addition causes a synchronous release of the reporter from the hook. Using the RUSH system, we analyzed different transport characteristics of various Golgi and plasma membrane reporters at physiological temperature in living cells. Using dual-color simultaneous live-cell imaging of two cargos, we observed intra- and post-Golgi segregation of cargo traffic, consistent with observation in other systems. We show preliminarily that the RUSH system is usable for automated screening. The system should help increase the understanding of the mechanisms of trafficking and enable screens for molecules that perturb pathological protein transport.

Structural Basis for Recruitment of Rab6-Interacting Protein 1 to Golgi via a RUN Domain

Small GTPase Rab6 regulates vesicle trafficking at the level of Golgi via recruitment of numerous and unrelated effectors. The crystal structure of Rab6a(GTP) in complex with a 378-residue internal fragment of the effector Rab6IP1 was solved at 3.2 angstroms resolution. This Rab6IP1 region encompasses an all alpha-helical RUN domain followed in tandem by a PLAT domain that adopts a beta sandwich fold. The structure reveals that the first and last alpha helices of the RUN domain mediate binding to switch I, switch II, and the interswitch region of Rab6. It represents the largest Rab-effector complex determined to date. Comparisons with the recent structure of Rab6 in complex with an unrelated effector, human golgin GCC185, reveals significant conformational changes in the conserved hydrophobic triad of Rab6. Flexibility in the switch and interswitch regions of Rab6 mediates recognition of compositionally distinct alpha-helical coiled coils, thereby contributing to Rab6 promiscuity in effector recruitment.

Identification and characterization of multiple novel Rab–myosin Va interactions

A systematic screen of the entire human Rab GTPase family for interactions with myosin Va identified 10 novel Rab partners for myosin Va, all of which belong to the endocytic recycling and post-Golgi secretory membrane network. However, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes.

Rab30 is required for the morphological integrity of the Golgi apparatus.

Rab GTPases are key coordinators of eukaryotic intracellular membrane trafficking. In their active states, Rabs localise to the cytoplasmic face of intracellular compartments where they regulate membrane trafficking processes. Many Rabs have been extensively characterised whereas others, such as Rab30, have to date received relatively little attention.

Golgi inheritance under a block of anterograde and retrograde traffic.

In mitosis, the Golgi complex is inherited following its dispersion, equal partitioning and reformation in each daughter cell. The state of Golgi membranes during mitosis is controversial, and the role of Golgi‐intersecting traffic in Golgi inheritance is unclear. We have used brefeldin A (BFA) to perturb Golgi‐intersecting membrane traffic at different stages of the cell cycle and followed by live cell imaging the fate of Golgi membranes in those conditions. We observed that addition of the drug on cells in prometaphase prevents mitotic Golgi dispersion. Under continuous treatment, Golgi fragments persist throughout mitosis and accumulate in a Golgi‐like structure at the end of mitosis. This structure localizes at microtubule minus ends and contains all classes of Golgi markers, but is not accessible to cargo from the endoplasmic reticulum or the plasma membrane because of the continuous BFA traffic block. However, it contains preaccumulated cargo, and intermixes with the reforming Golgi upon BFA washout. This structure also forms when BFA is added during metaphase, when the Golgi is not discernible by light microscopy. Together the data indicate that independent Golgi fragments that contain all classes of Golgi markers (and that can be isolated from other organelles by blocking anterograde and retrograde Golgi‐intersecting traffic) persist throughout mitosis.

Mapping the interactions between a RUN domain from DENND5/Rab6IP1 and sorting nexin 1.

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses.

C74, a new Rab6 partner, is also a centrosomic protein involved in microtubule nucleation

’ CNRSUMRJ I I, tJbversitt+ des Sciences et Technologitis de Lilte Hepatitis C virus (HCV) belongs to the FIaviviri&e family. Its genomr encodes two envelope glycoproteins (El and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which is retained in the endoplasmic reticuIum~ (ER) at steady state. Prolonged jnt+eractions of HCV glycoprotein complexes with ER chaperones has been observed and we have previously suggested that r+ssociation of HCV oligomers with calnexin, co&d be responsible for their continued retention in this compartment. However, more recent data indicate that native ElE2 complexes do not interact with calnexin and are still localized in the ER. HCV glycoprotein complexes may therefore contain an ER retention/retrieval signal which is probably important for the envelope formation. Indeed, for the flaviviruses, virions appear in intracellular vesicles (probably modified ER) and are released from-cells via the exocytosis pathway. To identify whether El and/or E2 contain an ERtargeting signal potentially involved in ER retention/retrieval of ElE2 complexes, these proteins were expressed alone and their intracellular localization studied. Due to misfolding of El irl the absence of EL?, no conch&n, on the localization of its native form, could be drawn from the expression of El alone. However, E2 expressed in the absence of El can fold properly and is retained in the ER, as shown by the lack of complex glycans, its intracellular distibudon and the absence of its expression on the cell surface. Replacement of the transmembrane domain of E2 with the anchor sequence of CD4 or a glycosyl phosphatidylinositol moiety was shown to be sufficient for its export to the cell surface. In addition, a chimeric protein containing the ectodomain of CD4 fused to the transmembrane domain of E2 was retained in the ER. These experiments indicate that the C-terminal 29 amino acids of JZ2 contain the information for its ER localization. We are also curre@y investigating whether the ER localization of ElE2 complex is duq to true retention or to retrieval from the cis-Golgi. Biochemical characterizstion of the glycans of HCV glycoproteins after metal&c labeling followed by long chases indicate that ER localization of HCV glycoprotein complex is due to true retention and not retrieval.