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	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.

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