Segregation of COPI-rich and anterograde-cargo-rich domains in endoplasmic-reticulum-to-Golgi transport complexes

AbstractMembrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in the selective packaging of anterograde cargo into coated transport vesicles budding from the ER [1]. In mammalian cells, these vesicles coalesce to form tubulo-vesicular transport complexes (TCs), which shuttle anterograde cargo from the ER to the Golgi complex [2–4]. In contrast, COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER [1,5–7]. The binding of COPI to COPII-coated TCs [3,8,9], however, has led to the proposal that COPI binds to TCs and specifically packages recycling proteins into retrograde vesicles for return to the ER [3,9]. To test this hypothesis, we tracked fluorescently tagged COPI and anterograde-transport markers simultaneously in living cells. COPI predominated on TCs shuttling anterograde cargo to the Golgi complex and was rarely observed on structures moving in directions consistent with retrograde transport. Furthermore, a progressive segregation of COPI-rich domains and anterograde-cargo-rich domains was observed in the TCs. This segregation and the directed motility of COPI-containing TCs were inhibited by antibodies that blocked COPI function. These observations, which are consistent with previous biochemical data [2,9], suggest a role for COPI within TCs en route to the Golgi complex. By sequestering retrograde cargo in the anterograde-directed TCs, COPI couples the sorting of ER recycling proteins [10] to the transport of anterograde cargo

Membrane targeting of palmitoylated Wnt and Hedgehog revealed by chemical probes

AbstractPalmitoylation of the Wnt and Hedgehog proteins is critical for maintaining their physiological functions. To date, there are no reported studies that characterize the cellular distribution of the palmitoylated forms of these proteins. Here, we describe the subcellular localization of palmitoylated Wnt and Sonic Hedgehog by using a highly sensitive and non-radioactive labeling method that utilizes alkynyl palmitic acid. We show that palmitoylated Wnt and Sonic Hedgehog localize to cellular membrane fractions only, highlighting a role for palmitoylation in the membrane association of these proteins. The method described herein has the utility to validate inhibitors of Wnt and Hedgehog acyltransferases in drug discovery, and enables further investigations of the role of palmitoylation in the secretion and signaling of these proteins

Membrane recruitment of coatomer and binding to dilysine signals are separate events

It has previously been shown that transport of newly synthesized proteins and the structure of the Golgi complex are affected in the Chinese hamster ovary cell line ldlF, which bears a temperature-sensitive mutation in the Coat protein I (COPI) subunit €-COP (Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-1224; Hobbie, L., Fisher, A. S., Lee, S., Flint, A., and Krieger, M. (1994) J. Biol. Chem. 269, 20958-20970). Here we pinpoint the site of the secretory block to an intermediate compartment between the endoplasmic reticulum (ER) and the Golgi complex and show that the distributions of ER-Golgi recycling proteins, such as KDEL receptor and p23, as well as resident Golgi proteins, such as mannosidase II, are accordingly affected. At the nonpermissive temperature, neither the stability of the COPI complex nor its recruitment to donor Golgi membranes is affected. However, the binding of coatomer to the dilysine-based ER-retrieval motif is impaired in the absence of €-COP, suggesting that dilysine signal binding is not the major means of COPI recruitment. Because expression of the exogenous chimera of €-COP and green fluorescent protein in ldlF cells at nonpermissive temperature rapidly restores the wild type properties, €-COP is likely to play an important role in the cargo selection events mediated COPI.</p

Kinetics of Hedgehog-Dependent Full-Length Gli3 Accumulation in Primary Cilia and Subsequent Degradation ▿ † ‡

Hedgehog (Hh) signaling in vertebrates depends on intraflagellar transport (IFT) within primary cilia. The Hh receptor Patched is found in cilia in the absence of Hh and is replaced by the signal transducer Smoothened within an hour of Hh stimulation. By generating antibodies capable of detecting endogenous pathway transcription factors Gli2 and Gli3, we monitored their kinetics of accumulation in cilia upon Hh stimulation. Localization occurs within minutes of Hh addition, making it the fastest reported readout of pathway activity, which permits more precise temporal and spatial localization of Hh signaling events. We show that the species of Gli3 that accumulates at cilium tips is full-length and likely not protein kinase A phosphorylated. We also confirmed that phosphorylation and βTrCP/Cul1 are required for endogenous Gli3 processing and that this is inhibited by Hh. Surprisingly, however, Hh-dependent inhibition of processing does not lead to accumulation of full-length Gli3, but instead renders it labile, leading to its proteasomal degradation via the SPOP/Cul3 complex. In fact, full-length Gli3 disappears with faster kinetics than the Gli3 repressor, the latter not requiring SPOP/Cul3 or βTrCP/Cul1. This may contribute to the increased Gli3 activator/repressor ratios found in IFT mutants

Anti-CD19 is internalized by dynamin-dependent, clathrin-mediated endocytosis and is delivered to lysosomes

<p><b>Copyright information:</b></p><p>Taken from "High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate"</p><p></p><p>British Journal of Haematology 2007;140(1):46-58.</p><p>Published online 07 Nov 2007</p><p>PMCID:PMC2228374.</p><p>© 2007 Genentech, Inc. Journal Compilation © 2007 Blackwell Publishing Ltd</p> (A) Ramos cells were pre-incubated for 30 min at 37°C with the following reagents: dimethyl sulphoxide (DMSO) (1); 1 μmol/l chlorpromazine (Cpmzn) (2), a clathrin-mediated endocytosis inhibitor; 80 μmol/l dynamin inhibitor dynasore, preincubated for 5 min only (3); 2 mmol/l methyl-β-cyclodextrin (MbC) (4) or 5 μg/ml filipin (5), both inhibitors of caveolar and lipid raft endocytosis. Alexa488-anti-CD19 (black bars) or Alexa488-transferrin (grey bars) were then added in the continuous presence of inhibitors for 30 min and surface quenched as in . Results were plotted as a percentage of uptake compared with the DMSO control and represent the average and standard deviation of three independent triplicate experiments. (B–D) Alexa488-anti-CD19 (green channel in B and D) was co-internalized with Alexa647-transferrin (shown in the red channel in C and D) in Ramos cells for 5 min, surface quenched with anti-Alexa488, fixed and imaged. (E–G) Alexa488-anti-CD19 (green channel in E and G) was chased for 3 h in Ramos cells in the presence of lysosomal protease inhibitors prior to fixation and staining with Alexa555-anti-LAMP1 (red channel in F and G). Yellow colour in the merged images in panels D and G indicates colocalization. Gamma levels were adjusted where necessary to better illustrate marker overlap. Arrows indicate examples of co-localized staining. Scale bar is 20 μm in the main panels and 6·7 μm in the 3×-magnified insets of the boxed region indicated in D