Transposition-based method for the rapid generation of gene-targeting vectors to produce Cre/Flp-modifiable conditional knock-out mice

Peer reviewe

Imaging and imagination: understanding the endo-lysosomal system

Lysosomes are specialized compartments for the degradation of endocytosed and intracellular material and essential regulators of cellular homeostasis. The importance of lysosomes is illustrated by the rapidly growing number of human disorders related to a defect in lysosomal functioning. Here, we review current insights in the mechanisms of lysosome biogenesis and protein sorting within the endo-lysosomal system. We present increasing evidence for the existence of parallel pathways for the delivery of newly synthesized lysosomal proteins directly from the trans-Golgi network (TGN) to the endo-lysosomal system. These pathways are either dependent or independent of mannose 6-phosphate receptors and likely involve multiple exits for lysosomal proteins from the TGN. In addition, we discuss the different endosomal intermediates and subdomains that are involved in sorting of endocytosed cargo. Throughout our review, we highlight some examples in the literature showing how imaging, especially electron microscopy, has made major contributions to our understanding of the endo-lysosomal system today

Cloning and characterization of Vear, a novel Golgi-associated protein involved in vesicle trafficking

Abstract The control and maintenance of the character, number and protein, carbohydrates and lipid composition of intracellular compartments in a changing environment is one of the fundamental features of a living cell. It is effected, to a large measure, by vesicular traffic which connects the various cellular compartments and handles the transportation of cargo between them. Movement of cargo occurs through a transport system in membrane-bounded containers called vesicles. Vesicles originate at the donor membrane from which they are transported to target organelles where they fuse with the acceptor membrane and deliver their cargo. At the donor site, cytosolic coat proteins or 'coats' bind to the donor membrane together with GTP (guanosine 5'-triphosphate)-binding regulatory proteins first to deform a bud, which is then pinched off as a coated vesicle. During budding and targeting events, a number of regulatory proteins interact with the coat components. Currently, several different coat proteins and their adaptor proteins are known. The purpose of this study was to characterize novel components participitating in intracellular vesicle transport. By using computer analysis and EST (expressed sequence tag) database searches, a previously unknown protein was found. Sequencing revealed the presence of a novel protein of 613 amino acids with a calculated molecular mass of 67,149 Da. Based on its structural features, possessing both a VHS domain and an "ear" domain, we named the protein Vear. With its VHS domain in its NH2 terminus, Vear shows similarity to several endocytosis-associated proteins. With the "ear" domain in its C-terminus, it resembles γ-adaptin, a heavy subunit of the AP-1 complex. Vear mRNA showed a widespread distribution in tissues, with high amounts of mRNA in the kidney, skeletal muscle, and cardiac muscle. At the subcellular level, Vear was localized to the Golgi complex in which it colocalized with the trans-Golgi marker γ-adaptin. The preferential membrane-association was demonstrated by subcellular fractionation in which Vear partitioned with the total membrane fraction. Golgi-associated subcellular localization for Vear was sensitive to a treatment with the fungal metabolite brefeldin A, suggesting an ARF (ADP-ribosylation factor)-dependent recruitment onto membranes. In transfection studies, the full-length Vear assembled on and caused structural "compaction" of the Golgi complex, while overexpression of the "ear" domain alone showed diffuse Golgi-localization without "compaction". The VHS domain, on the other hand, was mainly vesicle- and plasma membrane associated and did not show any association with Golgi. In skeletal muscle, Vear was detected preferentially in type I cells by immunohistochemistry and immunofluorescence microscopy. In normal kidney, Vear was exclusively present in glomerular epithelial cells (podocytes) and Vear protein was expressed in a developmentally regulated manner during glomerulogenesis. By immunolabeling electron microscopy, Vear was seen in vesicular and membrane structures adjacent to the Golgi complex. Vear was also abundant in the gastrointestinal tract in cells active in secretion. The results indicate that Vear is a novel vesicle transport-associated protein, detected mainly in the Golgi complex and localized in tissues in a highly cell-type specific manner

ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs and VPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function

Phosphoinositide-mediated clathrin adaptor progression at the trans-Golgi network

Clathrin coated vesicles mediate endocytosis and transport between the trans Golgi network (TGN) and endosomes in eukaryotic cells. Clathrin adaptors play central roles in coat assembly, interacting with clathrin, cargo, and membranes. Two major types of clathrin adaptors act in TGN-endosome traffic, Gga proteins and the AP-1 complex. Here we characterize the relationship between Gga proteins, AP-1, and other TGN clathrin adaptors using live cell and superresolution microscopy in yeast. We present evidence that Gga proteins and AP-1 are recruited sequentially in two waves of coat assembly at the TGN. Mutations that decrease phosphatidylinositol 4-phosphate (PI4P) levels at the TGN slow or uncouple AP-1 coat assembly from Gga coat assembly. Conversely, enhanced PI4P synthesis shortens the time between adaptor waves. Gga2p binds directly to the TGN PI4-kinase Pik1p and contributes to Pik1p recruitment. These results identify a PI4P-based mechanism for regulating progressive assembly of adaptor-specific clathrin coats at the TGN