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Role of xklp3, a subunit of the Xenopus kinesin II heterotrimeric complex, in membrane transport between the endoplasmic reticulum and the Golgi apparatus.

Le Bot N, Antony C, White J, Karsenti E, Vernos I - J. Cell Biol. (1998)

Bottom Line: A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus.The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain.Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Biophysics Program, European Molecular Biological Laboratory, D-69117 Heidelberg, Germany.

ABSTRACT
The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.

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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.
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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.

Mentions: To characterize Xklp3 we raised two polyclonal antibodies: one against the motor domain (anti–Xklp3-M) and another against the COOH-terminal tail domain (anti– Xklp3-tail). Both affinity-purified antibodies, as well as a monoclonal antibody, mAb X3T-3A6, recognized a single band with an apparent Mr of 95 kD on immunoblots of extracts from Xenopus eggs and from two different Xenopus cell lines, XL177 and A6 (Fig. 2 A). We then looked at Xklp3 ATP-dependent microtubule-binding activity. In egg extracts, Xklp3 copelleted with endogenous taxol-stabilized microtubules in the presence of 5′-adenylyllimidodiphosphate (AMP-PNP) and was released from the microtubule pellet by 10 mM ATP (Fig. 2 B), similar to the behavior of kinesin and other KLPs (Bloom and Endow, 1994).


Role of xklp3, a subunit of the Xenopus kinesin II heterotrimeric complex, in membrane transport between the endoplasmic reticulum and the Golgi apparatus.

Le Bot N, Antony C, White J, Karsenti E, Vernos I - J. Cell Biol. (1998)

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.
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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.
Mentions: To characterize Xklp3 we raised two polyclonal antibodies: one against the motor domain (anti–Xklp3-M) and another against the COOH-terminal tail domain (anti– Xklp3-tail). Both affinity-purified antibodies, as well as a monoclonal antibody, mAb X3T-3A6, recognized a single band with an apparent Mr of 95 kD on immunoblots of extracts from Xenopus eggs and from two different Xenopus cell lines, XL177 and A6 (Fig. 2 A). We then looked at Xklp3 ATP-dependent microtubule-binding activity. In egg extracts, Xklp3 copelleted with endogenous taxol-stabilized microtubules in the presence of 5′-adenylyllimidodiphosphate (AMP-PNP) and was released from the microtubule pellet by 10 mM ATP (Fig. 2 B), similar to the behavior of kinesin and other KLPs (Bloom and Endow, 1994).

Bottom Line: A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus.The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain.Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Biophysics Program, European Molecular Biological Laboratory, D-69117 Heidelberg, Germany.

ABSTRACT
The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.

Show MeSH
Related in: MedlinePlus