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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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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.
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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.

Mentions: The previous results pointed to a role for Xklp3 in the movement of tubular-vesicular elements between the ER and Golgi apparatus. To investigate the function of Xklp3, we transfected Xenopus cells with a plasmid expressing a truncated form of Xklp3 with either GFP (pEGFP– Xklp3-ST) or a hemagglutinin (HA) tag (pHA–Xklp3-ST) replacing the whole motor domain. Immunoprecipitation experiments with an anti-GFP antibody showed that the mutant protein could inhibit Kinesin II function by competing with the endogenous Xklp3 for the binding to the 85-kD subunit of the Kinesin II heterotrimeric complex (Fig. 1 B and Fig. 8 A). By immunofluorescence, both fusion proteins were mostly found distributed throughout the cytoplasm and not associated with the Golgi apparatus (Fig. 8, B and C). This could be explained in two ways: either the mutant protein could not be targeted to the vesicles to which the wild-type protein binds normally or the mutant protein prevented the localization of the Kinesin II complex to its target vesicles. In either case, this mutant clearly acted as a poison for the endogenous Kinesin II complex. Indeed, a similar construct has been used successfully to study Kinesin II dependent movement of the melanosomes in Xenopus melanophores (Tuma et al., 1998).


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)

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.
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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.
Mentions: The previous results pointed to a role for Xklp3 in the movement of tubular-vesicular elements between the ER and Golgi apparatus. To investigate the function of Xklp3, we transfected Xenopus cells with a plasmid expressing a truncated form of Xklp3 with either GFP (pEGFP– Xklp3-ST) or a hemagglutinin (HA) tag (pHA–Xklp3-ST) replacing the whole motor domain. Immunoprecipitation experiments with an anti-GFP antibody showed that the mutant protein could inhibit Kinesin II function by competing with the endogenous Xklp3 for the binding to the 85-kD subunit of the Kinesin II heterotrimeric complex (Fig. 1 B and Fig. 8 A). By immunofluorescence, both fusion proteins were mostly found distributed throughout the cytoplasm and not associated with the Golgi apparatus (Fig. 8, B and C). This could be explained in two ways: either the mutant protein could not be targeted to the vesicles to which the wild-type protein binds normally or the mutant protein prevented the localization of the Kinesin II complex to its target vesicles. In either case, this mutant clearly acted as a poison for the endogenous Kinesin II complex. Indeed, a similar construct has been used successfully to study Kinesin II dependent movement of the melanosomes in Xenopus melanophores (Tuma et al., 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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Related in: MedlinePlus