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First published online 25 January 2005
doi: 10.1242/jcs.01671

Journal of Cell Science 118, 723-732 (2005)
Published by The Company of Biologists 2005
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Protein kinase CK2 phosphorylates Sec63p to stimulate the assembly of the endoplasmic reticulum protein translocation apparatus

Xian Wang and Nils Johnsson*

Institut für Toxikologie und Genetik, Forschungszentrum Karlsruhe, Postfach 3640, 76021 Karlsruhe, Germany



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  		Fig. 1. The interaction between the C-terminus of Sec63p and the N-terminus of Sec62p is phosphorylation dependent.

(A) Representation of Sec63p and its C-terminal-derived fusion constructs F-Fpr1-63C47 and F-Fpr1-63C14. The sequence of the last 14 residues of Sec63p is given in the one-letter code. (B) Extracts of yeast cells expressing F-Fpr1-63C14 together with Sec62{Delta}C-125-Dha (lanes a, b, c) or alone (lanes d, e, f) were incubated with anti-Ha or anti-Flag antibodies to precipitate Sec62{Delta}C-125-Dha (lanes b, c) or F-Fpr1-63C14 (lanes e, f), respectively. Extracts (Ex), supernatant (Sn) and precipitates (P) were probed with anti-Flag antibody directly (lanes a-f) or after the treatment of the precipitate with calf intestinal phosphatase (CIP) (lane e). (C) In vivo phosphorylation of F-Fpr1-63C14. Yeast cells expressing F-Fpr1-63C14 (lanes a, b) or F-Fpr1-63C14T652; 654A (lane c) were labeled with [32P]O4. Extracts were incubated either with anti-Flag antibody (lanes b, c) or not (lane a) followed by the precipitation of the antibody, SDS-PAGE of the bound fraction, and autoradiography of the dried gel. Anti-Flag western blot of the immunoprecipitates of the unlabeled cells is shown in lanes d and e. (D) Sepharose-coupled F-Fpr1-63C14 was incubated without (–) or with (+) phosphatase (lanes a, b) followed by incubation with Sec62{Delta}C125-Dha (lane c). Precipitates and supernatants were subjected to 12.5% SDS-PAGE and immunoblot detection with anti-Flag (lanes a, b) or anti-Ha antibody (lanes c-g). LC indicates the light chain of the antibody.

 


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  		Fig. 2. The phosphorylatable threonines in position 652 and 654 of Sec63p are required for binding to the N-terminal domain of Sec63p. (A) Immunoprecipitation of F-Fpr1-63C14 (WT) and the mutants carrying single alanine exchanges in position 652 (T652A), 654 (T654A), 661 (S661A) or the exchange at position 652 and 654 (T652; 654A). The precipitates were either mock (–) or phosphatase (+) treated and subjected to anti-Flag detection after 12.5% SDS-PAGE. (B) Extracts of cells expressing F-Fpr1-63C14 (WT) or its mutants were anti-Flag immunoprecipitated and the precipitates were subsequently incubated with extracts containing Sec62{Delta}C125-Dha. Bound fractions were probed with anti-Flag (upper panel) or anti-Ha antibodies (lower panel) after 12.5% SDS-PAGE. An extract of cells expressing Sec62{Delta}C125-Dha is shown in lane a. (C) Sepharose-coupled F-Fpr1-63C47 was treated without or with phosphatase (– or +) and incubated with extracts containing Sec62{Delta}C125-Dha. The bound (lanes a-d) and unbound fractions (lanes e, f) were probed with anti-Flag (a, b) and anti-Ha antibodies (lanes c-f). (D) Same as C but with F-Fpr1-63C47 (WT), and its T/A mutants in position 652 and 654. LC and HC indicate the light and heavy chains of the antibody, respectively.

 


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  		Fig. 3. Full-length Sec63p is phosphorylated at its C-terminal domain in vivo. (A) A protein extract from wild-type cells was separated by 12.5% SDS-PAGE and transferred onto nitrocellulose. The blot was treated either with (+) or without (–) CIP and subsequently incubated with extracts from yeasts either expressing Sec62{Delta}C125-Dha (+) or not (–). Bound Sec62{Delta}C125-Dha was detected with anti-Ha antibody. (B) Extracts of yeast cells expressing Sec63p or its alleles were subjected to the Sec62{Delta}C125-Dha overlay assay (top), or detection with anti-Sec63p antibody (bottom). {Delta}C14 indicates a mutant of Sec63p lacking the last 14 amino acids.

 


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  		Fig. 6. Sec63p is phosphorylated by protein kinase CK2 in vivo and in vitro. (A) Yeast cells carrying a deletion of CKA1 and the indicated alleles of CKA2 were shifted to 37.5°C and the expression of F-Fpr1-63C14 was induced. Cell extracts were prepared at the indicated times and probed with anti-Flag antibody after transfer onto nitrocellulose. (B) As A but F-Fpr1-63C14 of cells grown for 3 hours at 37.5°C was precipitated by anti-Flag antibody and either mock treated (–) or treated with an unspecific phosphatase (+). (C) Autoradiogram (lanes a-e) of CIP-treated F-Fpr1-63C14 (WT) or F-Fpr1-63C14T652; 654A (T652; 654A) incubated in the presence (+) of [{gamma}-32P] GTP with Cka1-ProA attached to IgG-sepharose (CK2) or with mock-treated IgG-beads (–), or with (+) or without (–) heparin. Anti-Flag western blot of the substrates for the kinase assays (lanes f, g). (D) As A but with a CK2 and a ck2ts strain, that contain SEC63-ProA and expressing Sec63p ectopically from the PMET17-promotor. Cells were grown at 25°C in medium containing 10 mM methionine (+) and shifted to 37.5°C and methionine-free medium (–). Extracts were probed with the Sec62{Delta}C125-Dha overlay assay (upper panel) or with anti-Sec63p antibody (lower panel). (Lanes k and l) JD53 cells containing SEC63 (k) or SEC63-ProA (l) were probed with the Sec62{Delta}C125-Dha overlay assay (upper panel) or with anti-Sec63p antibody (lower panel).

 


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  		Fig. 4. Monitoring the effects of phosphorylation on the Sec63p-Sec62p interaction in vivo. (A) Extracts of yeast cells expressing Sec63-ProA or Sec63T654A-ProA were precipitated with IgG-sepharose and the supernatants (Sn) and the precipitates (P) were probed with anti-Sec62p serum. The precipitate was additionally probed with rabbit anti-goat/goat anti-rabbit antibodies. The asterisk (*) indicates cross-reacting bands of the anti-Sec62p serum. (B) Split-ubiquitin assay. Yeast cells coexpressing Sec63-Cub-RUra3p or its mutants together with Nug-Sec62p or Nug-Ubc6p were grown to an OD600 of 1. Four millilitres of these cultures and tenfold dilutions thereof were spotted on plates lacking uracil, tryptophan and histidine to select for the presence of the plasmids. To check for cell numbers dilutions were also spotted on SD+ura plates (not shown).

 


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  		Fig. 5. Yeast cells carrying alanine replacements of the phosphorylatable threonines in Sec63p display protein translocation defects. (A) Pulse analysis of CPY translocation. Wild-type cells (WT), cells carrying the alanine replacements in position 652 (T652A) or 654 (T654A) of Sec63p, or sec62-1 ts cells (sec62-1) were labeled with [35S]-methionine for 5 minutes and subjected to a CPY immunoprecipitation. P1 and P2 indicate the positions of the translocated and preproCPY the position of the nontranslocated fraction of CPY on the autoradiogram of the gel (Pilon et al., 1997Go). The shift in the apparent molecular weight after EndoH treatment (+) confirmed the localization of P1 and P2 in the secretory pathway. (B) Steady-state analysis. Yeast cells carrying the indicated alleles of SEC63 or SEC62 and expressing signal-sequence-bearing Cub-Ura3p constructs were spotted on plates without histidine and with or without uracil (+Uracil or –Uracil, respectively) to select for the presence of the plasmids. Growth of cells on SD-ura plates indicates a translocation defect for the respective signal sequence. (C) Synthetic lethality. Yeast cells containing SEC63 or sec63T654A and expressing Sec62p from a URA3 plasmid were transformed with the different TRP1 plasmids containing the indicated alleles of SEC62 carrying the Dha module at their C-termini (Wittke et al., 2000Go). Cells were plated on 5-FOA-containing media. Cells that contain the sec63T654A and the sec62{Delta}C35-DHA allele do not growth on this media, indicating a synthetic lethality between the two alleles.

 


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  		Fig. 7. Phosphorylated Sec63p is only slowly turned over. (A) Wild-type or cka2ts cells were shifted to the restrictive temperature, pulsed at the indicated times with [35S]-methionine and subjected to CPY immunoprecipitation (upper panel). Extracts of the same cells were also probed for the presence of the phosphorylated Sec63p by the Sec62{Delta}C125-Dha overlay assay (lower panel) (B) Summary. Newly made Sec63p is rapidly phosphorylated by protein kinase CK2. The phosphorylated Sec63p binds strongly to Sec62p and becomes protected against dephosphorylation. Certain conditions might induce a phosphatase activity that dephosphorylates Sec63p and thereby shifts the ratio from the bound to the free form.

 




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