Primary cilia are sensory structures involved in morphogen signalling during development, liquid flow in the kidney, mechanosensation, sight, and smell (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125–148; Singla, V., and J.F. Reiter. 2006. Science. 313:629–633.). Mutations that affect primary cilia are responsible for several diseases, including neural tube defects, polycystic kidney disease, retinal degeneration, and cancers (Badano et al., 2006; Singla and Reiter, 2006). Primary cilia formation and function requires tight integration of the microtubule cytoskeleton with membrane trafficking (Singla and Reiter, 2006), and this is poorly understood. We show that the Rab GTPase membrane trafficking regulators Rab8a, -17, and -23, and their cognate GTPase-activating proteins (GAPs), XM_037557, TBC1D7, and EVI5like, are involved in primary cilia formation. However, other human Rabs and GAPs are not. Additionally, Rab8a specifically interacts with cenexin/ODF2, a basal body and microtubule binding protein required for cilium biogenesis (Ishikawa, H., A. Kubo, S. Tsukita, and S. Tsukita. 2005. Nat. Cell Biol. 7:517–524), and is the sole Rab enriched at primary cilia. These findings provide a basis for understanding how specific membrane trafficking pathways cooperate with the microtubule cytoskeleton to give rise to the primary cilia.

The Golgi apparatus has long been suggested to be important for directing secretion to specific sites on the plasma membrane in response to extracellular signaling events. However, the mechanisms by which signaling events are coordinated with Golgi apparatus function remain poorly understood. Here, we identify a scaffolding function for the Golgi matrix protein GM130 that sheds light on how such signaling events may be regulated. We show that the mammalian Ste20 kinases YSK1 and MST4 target to the Golgi apparatus via the Golgi matrix protein GM130. In addition, GM130 binding activates these kinases by promoting autophosphorylation of a conserved threonine within the T-loop. Interference with YSK1 function perturbs perinuclear Golgi organization, cell migration, and invasion into type I collagen. A biochemical screen identifies 14-3-3ζ as a specific substrate for YSK1 that localizes to the Golgi apparatus, and potentially links YSK1 signaling at the Golgi apparatus with protein transport events, cell adhesion, and polarity complexes important for cell migration.

Rab-family GTPases are conserved regulators of membrane trafficking that cycle between inactive GDP-bound and activated GTP-bound states. A key determinant of Rab function is the lifetime of the GTP-bound state. As Rabs have a low intrinsic rate of GTP hydrolysis, this process is under the control of GTP-hydrolysis-activating proteins (GAPs). Due to the large number of Rabs and GAPs that are encoded by the human genome, it has proven difficult to assign specific functional relationships to these proteins. Here, we identify a Rab5-specific GAP (RabGAP-5), and show that RN-Tre (previously described as a Rab5 GAP) acts on Rab41. RabGAP-5 overexpression triggers a loss of the Rab5 effector EEA1 from endosomes and blocks endocytic trafficking. By contrast, depletion of RabGAP-5 results in increased endosome size, more endosome-associated EEA1, and disrupts the trafficking of EGF and LAMP1. RabGAP-5 therefore limits the amount of activated Rab5, and thereby regulates trafficking through endosomes.

Rab GTPases control vesicle movement and tethering membrane events in membrane trafficking. We used the 38 human Rab GTPase activating proteins (GAPs) to identify which of the 60 Rabs encoded in the human genome function at the Golgi complex. Surprisingly, this screen identified only two GAPs, RN-tre and TBC1D20, disrupting both Golgi organization and protein transport. RN-tre is the GAP for Rab43, and controls retrograde transport into the Golgi from the endocytic pathway. TBC1D20 is the ER-localized GAP for Rab1, and is the only GAP blocking the delivery of secretory cargo from the ER to the cell surface. Strikingly, its expression causes the loss of the Golgi complex, highlighting the importance of Rab1 for Golgi biogenesis. These effects can be antagonized by reticulon, a binding partner for TBC1D20 in the ER. Together, these findings indicate that Rab1 and Rab43 are key Rabs required for the biogenesis and maintenance of a functional Golgi structure, and suggest that other Rabs acting at the Golgi complex are likely to be functionally redundant.

In this review we will focus on the recent advances in how coiled-coil proteins of the golgin family give identity and structure to the Golgi apparatus in animal cells. A number of recent studies reveal a common theme for the targeting of golgins containing the ARL-binding GRIP domain, and the related ARF-binding GRAB domain. Similarly, other golgins such as the vesicle tethering factor p115 and Bicaudal-D are targeted by the Rab GTPases, Rab1 and Rab6, respectively. Together golgins and their regulatory GTPases form a complex network, commonly known as the Golgi matrix, which organizes Golgi membranes and regulates membrane trafficking.

Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A–C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.

GCC185, a trans-Golgi network-localized protein predicted to assume a long, coiled-coil structure, is required for Rab9-dependent recycling of mannose 6-phosphate receptors (MPRs) to the Golgi and for microtubule nucleation at the Golgi via CLASP proteins. GCC185 localizes to the Golgi by cooperative interaction with Rab6 and Arl1 GTPases at adjacent sites near its C terminus. We show here by yeast two-hybrid and direct biochemical tests that GCC185 contains at least four additional binding sites for as many as 14 different Rab GTPases across its entire length. A central coiled-coil domain contains a specific Rab9 binding site, and functional assays indicate that this domain is important for MPR recycling to the Golgi complex. N-Terminal coiled-coils are also required for GCC185 function as determined by plasmid rescue after GCC185 depletion by using small interfering RNA in cultured cells. Golgi-Rab binding sites may permit GCC185 to contribute to stacking and lateral interactions of Golgi cisternae as well as help it function as a vesicle tether. INTRODUCTIONThe Golgi complex plays a central role in processing and sorting of secreted and membrane proteins. In mammalian cells, it is made up of a set of flattened cisternae that are organized as a stack (Farquhar and Palade, 1998). Within the Golgi, proteins undergo oligosaccharide side chain maturation, and they are sorted to different destinations, including apical or basolateral plasma membranes, prelysosomes, or secretory storage granules, as they exit this compartment.The Golgi surface is decorated with proteins called Golgins (Short et al., 2005;Sztul and Lupashin, 2006). Golgins are characterized by their predicted, highly extended, coiled-coil structures, and they typically have a high glutamic acid content (Ramirez et al., 2008). Certain Golgins have been implicated as transport vesicle tethers that help capture vesicles and bring them closer to their cognate targets; they are also essential for the maintenance of normal Golgi structure. For example, temperature-sensitive cells lacking GM130 have a disrupted Golgi at the nonpermissive temperature (Vasile et al., 2003); loss of Golgin-84 (Diao et al., 2003), Golgin-245 (Yoshino et al., 2005), TATA modulatory factor (Fridmann-Sirkis et al., 2004), GRASP55 (Feinstein and Linstedt, 2008), GRASP65 (Puthenveedu et al., 2006), and the so-called COG complex (Zolov and Lupashin, 2005) all lead to disruption of the Golgi ribbon. Thus, the Golgi complex is highly sensitive to the loss of any one of a large collection of Golgi-associated proteins.Most Golgins are peripherally associated with this organelle by virtue of interaction with small GTPases of the Rab and/or Arf (Arl) families; a few contain C-terminal membrane anchors (Short et al., 2005). Four human Golgins contain a so-called GRIP domain at their C termini that enables them to bind the Arl1 GTPase at the trans-Golgi network (TGN); Arl1 is essential for the Golgi targeting of multiple GRIP domain-containing proteins (Lu and Hong, 2003;Panic et al., 2003...

Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 2∶1 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.dsFv | imaging | immunotoxin | protein engineering