Le Bot N , Antony C , White J , Karsenti E , Vernos I .

	Figure 2. Xklp3 is a subunit of the Kinesin II heterotrimeric complex and is associated to Golgi membranes. (A) Characterization of antibodies against Xklp3. Antibodies raised against the NH2-terminal (α-Motor) and the COOH-terminal (α-Tail) region of Xklp3 recognize a single band of ∼95 kD on Western blots of Xenopus low-speed egg extract, XL177, and A6 cell extracts. The monoclonal anti-tail, mAb X3T-3A6, recognizes a single band at 95 kD in low-speed egg extract. (B) Xklp3 cosediment with microtubules in the presence of AMP-PNP, and is released from the MT by addition of ATP. Xenopus egg extract (High Speed Extract) was used to prepare a fraction enriched in motor proteins (Materials and Methods). The high speed extract, the MT pellet in presence of AMP-PNP, the motor proteins released from MT by ATP, and the MT pellet after ATP elution were analyzed by Western blot and probed with the anti–Xklp3-tail antibody. Most of Xklp3 is released from the microtubules by 10 mM ATP. (C and D) Xklp3 is a subunit of a heterotrimeric complex, homologous to the Kinesin II complex. (C) Coomassie-stained gel of an immunoprecipitation from low-speed Xenopus egg extract using the anti–Xklp3-tail antibody. Two proteins (80 and 110 kD, arrows) coimmunoprecipitate with Xklp3 (asterisk). A control was done using protein A beads alone (Mock lane), similar results were obtained using nonspecific rabbit IgG. Arrowhead, lower bands that represent the antibodies' heavy chains. (D) Western Blot of the immunoprecipitated fraction. The 95-kD band is recognized by the anti–Xklp3-tail antibody (α-Xklp3). The 85-kD band is recognized by an antibody against SpKRP85, the second KLP subunit of Kinesin II (α-KRP85), and the 110-kD band by an antibody against SpKAP115, the third nonmotor subunit (α-KAP115). (E) Double immunofluorescence on Xenopus XL177 cells with the anti–Xklp3-tail (left panel, green) and an anti-tubulin antibody (middle panel, red). Xklp3 localizes to an extended juxtanuclear network. The nuclear pattern observed here is not representative of Xklp3 staining (see other figures where it is not present). Bar, 10 μm.
	Figure 3. Xklp3 colocalization with different Golgi markers. (A) Double immunofluorescence on XL177 cells with the anti– Xklp3-tail antibody and antibodies against different markers for the Golgi apparatus, as indicated. Xklp3 colocalizes partially with the lectin HP and completely with the KDEL-receptor. (B) A6 cells stably expressing GalT–GFP or GalNacT2–GFP were fixed and processed for immunofluorescence with the anti–Xklp3-tail antibody. Xklp3 colocalizes with both markers. Red, Xklp3; green, corresponding marker. Bars, 10 μm.
	Figure 4. Xklp3 does not colocalize with ER the PDI or with post-Golgi structures en route to the plasma membrane. (Top) XL177 cells stained with the anti–Xklp3-tail antibody and an anti-KDEL signal antibody (anti-PDI). (Bottom) A6 cells transiently transfected with VSV-G–GFP and stained with the anti–Xklp3-tail antibody. Xklp3 colocalizes with VSV-G–GFP in the Golgi apparatus but not in structures at the vicinity of the plasma membrane, thus indicating that Xklp3 is not associated with post-Golgi membranes. Red, Xklp3; green, corresponding marker. Bar, 10 μm.
	Figure 5. Xklp3 and the KDEL receptor have a similar behavior upon BFA treatment. (A) A6 cells expressing Gal NacT2–GFP constitutively were incubated with 5 αg/ml of BFA for 5 min or 1 h and then stained with the anti– Xklp3-tail antibodies. After 5 min of BFA treatment, GalNacT2–GFP starts to be redistributed into the ER often appearing on tubular structures. The anti–Xklp3-tail antibody gives a punctuate staining dispersed in the cytoplasm, often associated to but not overlapping with the GalNacT2–GFP staining (enlarged region). After 15 min, the structures stained by GalNacT2–GFP and the vesicular structures stained with the anti–Xklp3-tail antibody are completely distinct (1 hour, enlarged region). (B) XL177 cells were incubated with 5 μg/ml of BFA for 5 min or 1 h and double stained with the anti–Xklp3-tail and the anti–KDEL-receptor antibodies. The KDEL-receptor and Xklp3 colocalize on vesicular structures at early time points (enlarged region, overlay, 5 min). After 1 h of treatment, vesicular structures positive for both Xklp3 and the KDEL-receptor are still seen, but part of this colocalization is lost (enlarged region, overlay, 1 hour). Red, Xklp3; green, corresponding marker. Bar, 10 μm.
	Figure 6. Xklp3 localizes to tubular-vesicular structures between the ER and the Golgi. (A) Double immunogold labeling of Xklp3 (mAb X3T-3A6, 10-nm gold) and the KDEL-receptor (polyclonal anti-erd2, 5-nm gold) on cryosections from A6-GalT–GFP cells. Xklp3 is present on tubular-vesicular structures (arrows and arrowheads) around the Golgi-cisternae (G) where it colocalizes with the KDEL-receptor (arrows). Similar results are obtained with the polyclonal anti-tail (see Table I for quantification). (B) Double immunogold labeling of Xklp3 (monoclonal anti–Xklp3-tail, mAb X3T-3A6, 10-nm gold) and the GalT–GFP (polyclonal anti-GFP, 5-nm gold). GalT–GFP is localized exclusively to the Golgi cisternae and there is no clear colocalization with Xklp3 (arrowheads) When Xklp3 is found on the Golgi stacks, it is always at its periphery, on budding-like structures (A–C). Similar results are obtained with the polyclonal anti-tail (see Table I for quantification). (C) Double immunogold labeling of Xklp3 (polyclonal anti–Xklp3-tail, 10-nm gold, arrows and arrowheads) and PDI (monoclonal anti-PDI, 5-nm gold). The PDI is found in cisternae throughout the cytoplasm. Xklp3 is often seen on one side of these ER membrane structures (arrows) (see Table I for quantification). Bar, 100 nm.
	Figure 7. Xklp3 is associated to heavy membranes. (A) Xklp3 is found in a membrane fraction prepared from egg extracts. Western blot of a high-speed cytosol and the corresponding membrane fraction with the anti–Xklp3-tail antibody. Equivalent volumes from each fraction were loaded on each lane. (B) The membrane fraction shown in A was resuspended in 2 M sucrose, loaded at the bottom of a sucrose step gradient, and then centrifuged to equilibrium. Equivalent volumes of the different fractions were loaded on PAGE and transferred to nitrocellulose. The Western blot was probed with the anti–Xklp3-tail antibody. (1) 2 M/1.3 M sucrose (Bottom); (2) 1.3 M/0.86 M (heavy membranes, ∼ER); (3) 0.86 M/0.5 M (membranes ∼heavy Golgi); (4) 0.5 M/0.25 M (light membranes). (C) A fraction of Xklp3 is associated to heavy membranes from A6 cells extract. Cytosol (PNS) was prepared from A6 cells expressing GFP–mannosidase II constitutively as described in Materials and Methods. The PNS was then loaded on a sucrose step gradient (sucrose concentrations are written above fraction numbers; arrows, interface between two sucrose concentrations). Fractions were collected and run on PAGE. The Western blot was probed sequentially with the following antibodies: anti–Xklp3-tail, anti-PDI (as an ER marker), anti-GFP (to detect the Golgi marker mannosidase II–GFP), and anti-tubulin (as a nonmembrane-associated molecule). Xklp3 is present in soluble fractions 1–4. In addition, there are two peaks of Xklp3 in fractions where heavy membranes sediment: one around fraction 7 corresponding to membranes enriched in mannosidase II–GFP, and another around fraction 9–10 corresponding to membranes enriched in PDI.
	Figure 8. Effect of the expression of Xklp3–ST on Golgi staining by HP lectin. (A) The Xklp3–ST construct is able to bind to the endogenous Xklp3– KLP partner. A6 cells were transiently transfected with GFP–Xklp3-ST. The cytosol was used for immunoprecipitation with an anti-GFP antibody (lanes GFP–Xklp3-ST). The immunoprecipitate was probed with the anti-GFP and the anti-KRP85 antibodies. As a control we repeated this experiment with nontransfected cells (lanes None). Lane E is the crude extract fraction and lane IP the immunoprecipitate (loaded five times more than the extract). In transfected cells the anti-GFP antibody immunoprecipitates GFP–Xklp3-ST and a protein recognized by the anti-KRP85 antibody, probably the KLP partner for Xklp3. (B) Expression of Xklp3–ST has no effect on microtubules. A6 cells transiently transfected with GFP–Xklp3-ST were fixed and processed for immunofluorescence with an anti-tubulin antibody. Cells expressing Xklp3-ST have a normal microtubule network focused at the MTOC. (C) Suppression of Golgi staining by the lectin from Helix pomatia in cells expressing Xklp3–ST. A6 cells were transiently transfected with GFP–Xklp3-ST (asterisks), fixed, and then stained with rhodamine-labeled HP lectin. A projection of six sections taken with a LSCM is shown. In cells transfected with Xklp3–ST, the lectin no longer recognizes any identifiable Golgi structure. In contrast, the Golgi apparatus of non transfected cells is strongly stained. As a control we transfected A6 cells with GFP–Xklp1 (a Xenopus mitotic motor, asterisk). In these cells, the Golgi apparatus was normally stained by the HP lectin (bottom). (D) Quantification of the defective Golgi staining by the HP lectin. A6 cells were transiently transfected by GFP–Xklp3-ST, HA–Xklp3-ST, or GFP–Xklp1. 50–100 transfected cells were counted for each experiment. Bars correspond to the percentage of cells in each category. The standard deviations are shown (n = 4 for GFP– Xklp3-ST, HA–Xklp3-ST and n = 1 for GFP–Xklp1). White bars, normal Golgi HP lectin staining; black bars, abnormal or lack of staining. Note that 60–70% of Xklp3-ST–transfected cells have an abnormal Golgi staining, whereas this happens only in 10% of GFP-Xklp1–transfected cells. (E) The lack of Golgi staining by the HP lectin in cells expressing Xklp3–ST may reflect a defective transport from the ER to the Golgi. A6 cells expressing GalNacT2–GFP constitutively were transiently transfected with HA–Xklp3-ST (asterisks). Cells were fixed and double stained with a polyclonal anti-HA antibody, then visualized by an AMCA conjugated anti-rabbit antibody, and with rhodamine-labeled HP lectin. Left, red, HA–Xklp3-ST; green, GalNacT2–GFP. Right, HP lectin staining. The Golgi apparatus appears normal but is not stained by the rhodamine-labeled HP lectin. (F) In A6 cells, the lectin from HP binds to proteins en route along the secretory pathway. A6 cells expressing GalNacT2–GFP constitutively were treated for 3 h with cycloheximide, fixed, and then stained with rhodamine-labeled HP lectin. As above, the fluorescent lectin does not stain the Golgi apparatus. Bars, 10 μm.
	Figure 9. Newly synthesized GalNacT2–GFP and GalT–GFP proteins do not reach the Golgi apparatus in cells expressing Xklp3–ST. (A) A6 cells were transiently cotransfected with HA–Xklp3-ST and GalNacT2–GFP. Transfected cells (asterisks) were detected by immunofluorescence and confocal series were taken. A projection of six sections is presented. In cotransfected cells, GalNacT2–GFP is seen in aggregates in the cytoplasm and in the ER. In control cells, cotransfected with Xklp1-ST–myc and GalNacT2–GFP, the GFP marker localized to the Golgi apparatus. Bar, 10 μm. (B) Quantification of the defect of Golgi–GFP markers localization. A6 cells were transiently transfected with the different combinations indicated. Cells were scored as either having the GFP Golgi marker localized normally to the Golgi, to the ER and Golgi, or to the ER and aggregates. Bars show the percentage of cells in each category and the error bars show the standard deviation (n = 3). (C) Cells transiently cotransfected with HA–Xklp3-ST and GalNacT2–GFP exhibit an abnormal lectin staining. GalNacT2-GFP–expressing cells are detected by the GFP tag fluorescence. Cells were double labeled with a rabbit anti-HA antibody (then visualized by an AMCA conjugated anti-rabbit antibody) and the fluorescent lectin HP. Single confocal planes are presented. Left, note that two cells transfected only with GalNacT2–GFP (asterisks) show normal labeling of the Golgi by the HP lectin. Cotransfected cells show GalNacT2–GFP either in ER and Golgi (left panel, arrow 1) or in ER and aggregates (left panel, arrow 2; right panel). ∫Bar, 10 μm.

Arnheiter, Direct visualization of protein transport and processing in the living cell by microinjection of specific antibodies. 1984, Pubmed

Arnheiter, Direct visualization of protein transport and processing in the living cell by microinjection of specific antibodies. 1984, Pubmed
Boleti, Xklp2, a novel Xenopus centrosomal kinesin-like protein required for centrosome separation during mitosis. 1996, Pubmed , Xenbase
Burkhardt, Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution. 1997, Pubmed
Cole, Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. 1998, Pubmed
Cole, Novel heterotrimeric kinesin-related protein purified from sea urchin eggs. 1993, Pubmed , Xenbase
Corthésy-Theulaz, Cytoplasmic dynein participates in the centrosomal localization of the Golgi complex. 1992, Pubmed
Doms, Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. 1989, Pubmed
Dorner, Characterization of KIF1C, a new kinesin-like protein involved in vesicle transport from the Golgi apparatus to the endoplasmic reticulum. 1998, Pubmed
Echard, Interaction of a Golgi-associated kinesin-like protein with Rab6. 1998, Pubmed
Feiguin, Kinesin-mediated organelle translocation revealed by specific cellular manipulations. 1994, Pubmed
Füllekrug, Characterization of brefeldin A induced vesicular structures containing cycling proteins of the intermediate compartment/cis-Golgi network. 1997, Pubmed
Griffiths, Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells. 1994, Pubmed
Harada, Golgi vesiculation and lysosome dispersion in cells lacking cytoplasmic dynein. 1998, Pubmed
Henson, Immunolocalization of the heterotrimeric kinesin-related protein KRP(85/95) in the mitotic apparatus of sea urchin embryos. 1995, Pubmed
Kondo, KIF3A is a new microtubule-based anterograde motor in the nerve axon. 1994, Pubmed
Kozminski, The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane. 1995, Pubmed
Kreis, Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. 1986, Pubmed
Lewis, Ligand-induced redistribution of a human KDEL receptor from the Golgi complex to the endoplasmic reticulum. 1992, Pubmed
Liou, Improving structural integrity of cryosections for immunogold labeling. 1996, Pubmed
Lippincott-Schwartz, Cytoskeletal proteins and Golgi dynamics. 1998, Pubmed
Lippincott-Schwartz, Unravelling Golgi membrane traffic with green fluorescent protein chimeras. 1998, Pubmed
Lippincott-Schwartz, Kinesin is the motor for microtubule-mediated Golgi-to-ER membrane traffic. 1995, Pubmed
Lupas, Predicting coiled coils from protein sequences. 1991, Pubmed
Miller, Comparison of in vivo and in vitro ribosomal RNA synthesis in nucleolar mutants of Xenopus laevis. 1977, Pubmed , Xenbase
Morris, Heterotrimeric kinesin-II is required for the assembly of motile 9+2 ciliary axonemes on sea urchin embryos. 1997, Pubmed
Nilsson, Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum. 1989, Pubmed
Pavelka, Localization of binding sites for concanavalin A, Ricinus communis I and Helix pomatia lectin in the Golgi apparatus of rat small intestinal absorptive cells. 1985, Pubmed
Pazour, A dynein light chain is essential for the retrograde particle movement of intraflagellar transport (IFT). 1998, Pubmed
Pesavento, Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport. 1994, Pubmed
Presley, ER-to-Golgi transport visualized in living cells. 1997, Pubmed
Rafferty, The length of secondary chromosomal constrictions in normal individuals and in a nucleolar mutant of Xenopus laevis. 1969, Pubmed , Xenbase
Rashid, Heterodimerization of the two motor subunits of the heterotrimeric kinesin, KRP85/95. 1995, Pubmed
Reinsch, Movement of nuclei along microtubules in Xenopus egg extracts. 1997, Pubmed , Xenbase
Rogers, Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro. 1997, Pubmed , Xenbase
Roth, Cytochemical localization of terminal N-acetyl-D-galactosamine residues in cellular compartments of intestinal goblet cells: implications for the topology of O-glycosylation. 1984, Pubmed
Scheel, Dissociation of coatomer from membranes is required for brefeldin A-induced transfer of Golgi enzymes to the endoplasmic reticulum. 1997, Pubmed
Scholey, Kinesin-II, a membrane traffic motor in axons, axonemes, and spindles. 1996, Pubmed
Sciaky, Golgi tubule traffic and the effects of brefeldin A visualized in living cells. 1997, Pubmed
Shima, Partitioning of the Golgi apparatus during mitosis in living HeLa cells. 1997, Pubmed
Shimizu, SMAP, an Smg GDS-associating protein having arm repeats and phosphorylated by Src tyrosine kinase. 1996, Pubmed
Slot, Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. 1991, Pubmed
Smith, Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. 1988, Pubmed
Tabish, Exclusive expression of C. elegans osm-3 kinesin gene in chemosensory neurons open to the external environment. 1995, Pubmed
Tang, Molecular cloning, characterization, subcellular localization and dynamics of p23, the mammalian KDEL receptor. 1993, Pubmed
Tang, Differential response of resident proteins and cycling proteins of the Golgi to brefeldin A. 1995, Pubmed
Tooze, Condensation-sorting events in the rough endoplasmic reticulum of exocrine pancreatic cells. 1989, Pubmed
Tuma, Heterotrimeric kinesin II is the microtubule motor protein responsible for pigment dispersion in Xenopus melanophores. 1998, Pubmed , Xenbase
Vashishtha, The kinesin-homologous protein encoded by the Chlamydomonas FLA10 gene is associated with basal bodies and centrioles. 1996, Pubmed , Xenbase
Vernos, Multiple kinesin-like transcripts in Xenopus oocytes. 1993, Pubmed , Xenbase
Vernos, Xklp1, a chromosomal Xenopus kinesin-like protein essential for spindle organization and chromosome positioning. 1995, Pubmed , Xenbase
Walczak, XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly. 1996, Pubmed , Xenbase
Wedaman, Sequence and submolecular localization of the 115-kD accessory subunit of the heterotrimeric kinesin-II (KRP85/95) complex. 1996, Pubmed
Yamazaki, Cloning and characterization of KAP3: a novel kinesin superfamily-associated protein of KIF3A/3B. 1996, Pubmed
Yamazaki, KIF3A/B: a heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed motor for membrane organelle transport. 1995, Pubmed
Yang, Characterization of the KIF3C neural kinesin-like motor from mouse. 1998, Pubmed