Abstract

Arc35p, a component of the Arp2/3 complex, plays at least two distinct roles, regulating the actin cytoskeleton, but also microtubule function during cell division. Both functions involve calmodulin (CMD1). To investigate the pathway affecting microtubule function, we identified genes that are able to suppress the temperature-sensitive growth defect of the arc35-1 strain. Genes encoding γ-tubulin (TUB4) or any subunit of casein kinase II (CKII) suppressed this growth defect, but did not suppress the growth defect of a mutant in another subunit of the Arp2/3 complex, arp2-1. We could also show a physical association of Arc35p with subunits of CKII, Cmd1p, and Tub4p. Based on the exclusive localization of Arc35p to the cytosolic Arp2/3 complex and on mutant phenotypes, we propose that the role of the Arc35p/CKII interaction might be to activate a cytosolic pool of γ-tubulin, likely via calmodulin, for its nuclear and/or cytoplasmic functions.

Introduction

The Arp2/3 complex is a conserved multisubunit complex comprising, among others, the actin-related proteins Arp2 and Arp3 [1,2]. Subunits of the complex have been co-localized with filamentous actin to highly dynamic regions of the cell cortex [3–7]. Analysis of yeast strains bearing temperature-sensitive (ts) alleles of the actin-related proteins, arp2 and arp3, indicates an involvement of the complex in organization of the actin cytoskeleton, endocytosis, and in integrity and mobility of the actin patches [7–9]. A central role for the complex in organizing the actin cytoskeleton is also supported by in vitro findings that the complex caps the pointed ends of actin filaments and nucleates new filaments from the sides of preexisting filaments [10,11].

Recent experiments in yeast, however, indicate that the function of the Arp2/3 complex may not be restricted to its involvement in nucleation and organization of actin filaments. First, Arp2p has been localized to the nucleus where it seems to regulate nuclear import [12]. Second, mutations in ARC35, the 35 kDa component of the complex, lead to genetically separable defects in the actin and microtubule cytoskeletons, both of which can be suppressed by overexpression of calmodulin [13,14].

In Saccharomyces cerevisiae, calmodulin is encoded by a single essential gene, CMD1[15]. Systematic mutational analysis revealed at least four essential functions for Cmd1p [16]. One function of calmodulin in yeast is an involvement in mitosis: certain ts mutants display either defects in mitotic spindle formation/elongation [16] or in maintenance of spindle integrity [17,18]. Experiments with mammalian cells support an involvement of calmodulin in mitosis [19–21], even though the precise role is unclear.

Calmodulin was found to be an in vitro target of mammalian casein kinase II (CKII) [22,23]. Yeast CKII consists of two catalytic subunits, Cka1p and Cka2p, and two regulatory subunits, Ckb1p and Ckb2p [24–27]. CKII activity is essential in yeast [25]. Analysis of ts alleles of the yeast catalytic subunits has suggested that phosphorylation by CKII is required during multiple stages of the cell cycle, including mitosis.

In this study, we provide further insight into the regulation of the tubulin cytoskeleton by Arc35p. CKII was isolated as a suppressor of the cell cycle, but not the endocytic defect of arc35-1. We demonstrate genetic and biochemical interactions between Arc35p, Cmd1p, CKII, and Tub4p. Based on localization and genetic interaction data, we propose a model in which Arc35p as a subunit of the Arp2/3 complex, Cmd1p, and CKII might regulate some aspect of the Tub4p mitotic metaphase function.

Materials and methods

Yeast media, genetic techniques, and yeast strains

Strains without plasmids were grown in complete medium YPUADT (2% glucose, 2% peptone, 1% yeast extract, 40 μg ml−1 uracil, 40 μg ml−1 adenine, and 40 μg ml−1 tryptophan, 2% agar for solid medium). Strains bearing plasmids were selected on yeast nitrogen base minimal medium containing the required nutritional supplements [28]. Sporulation, tetrad dissection, and scoring of genetic markers were performed as described [28]. Recombinant lyticase was purified from Escherichia coli[29]. Transformation of yeast cells was accomplished by the lithium acetate method [30].

All yeast strains used in this study are listed in Table 1. Strain RH3428 was generated from RH3431 by sporulation, tetrad dissection, and scoring of appropriate genetic markers. Strain RH4547 was generated from RH2887 by replacing one genomic copy of CKA2 with the selectable marker kanMX4. The deletion cassette was amplified from pFA6a-kanMX4[31] with the oligonucleotides CKA2Δ_1 (5′-AAT AGA AGG AAC AAT AAA CCT AAA AGA ATA GAA GAA ACA GAC GTA CGC TGC AGG TCG AC) and CKA2Δ_2 (5′-TAC ATT CGT TGT TAA ATA GAG TTT GCA ATT TAA TAT TCA CAA TCG ATG AAT TCG AGC TC). Homology to CKA2 is indicated in bold, homology to pFA6a-kanMX4 in italic. Transformants were selected on geneticin-containing medium. RH4551 was generated by transforming RH4547 with plasmid pRS316::cka2-13::5MYC::6HIS and by subsequent replacement of the genomic copy of CKA1 with the selectable marker HIS3MX6. The deletion cassette was amplified from pFA6a-HIS3MX6[32] with the oligonucleotides CKA1Δ_1 (5′-ACA AAA ATA GGG GGT TGT AGA AGG AAT ATT TGA TTC GAA CTC GTA CGC TGC AGG TCG AC) and CKA1Δ_2 (5′-CAG ATG GTA AAA AAA AGT AAT CGT TAT ATC GTT TGT CAG TGA TCG ATG AAT TCG AGC TC). Homology to CKA1 is indicated in bold, homology to pFA6a-HIS3MX6 in italic. For the construction of RH4552, RH4551 was transformed with plasmid YCplac22::cka1-13::3HA::6HIS. Control for loss of plasmid pRS316::cka2-13::5MYC::6HIS was done on plates containing 5-fluoroorotic acid [28]. Strains RH4841 and RH4845 were generated by crossing RH4552 or RH4551, respectively, to RH2884. Strains DBY7445, DBY7449, and DBY7443 were crossed with RH2884 to produce RH4848, RH4851, and RH4852. For the construction of RH4839, RH2877 was transformed with a 3HA::kanMX4 cassette for C-terminal tagging of HHF2[33] amplified with the following oligonucleotides using pFA6a-3HA-kanMX4 as a template: H4-c-fus5′ (5′-GCT TTG AAG AGA CAA GGT AGA ACC TTA TAT GGT TTC GGT GGTGGT CGA CGG ATC CCC GGG) and H4-c-fus3′ (5′-CAT ACA TAA GGT TCT ATT ATA TTC CCA ATA GAA TGA TCG TTACAT CGA TGA ATT CGA GCT CG). Homology to HHF2 is shown in bold, homology to pFA6a-3HA-kanMX4 in italic.

1

Yeast strains

Strain Relevant markers Reference 
RH2877 Mata ade2 leu2 lys2 trp1 ura3 bar1 Riezman collection 
RH2884 Matα ade2 his3 leu2 trp1 ura3 bar1 Riezman collection 
RH2887 Mata his3 leu2 lys2 trp1 ura3 bar1 Riezman collection 
RH3428 Mata arc35-1 ade2 leu2 trp1 ura3 bar1 this study 
RH3429 Mata arc35-1 ade2 his4 leu2 lys2 ura3 bar1 [13
RH3431 Mata/Matα arc35-1/ARC35 ade2/ADE2 his4/HIS4 leu2/leu2 lys2/LYS2 trp1/TRP1 ura3/ura3 bar1/bar1 [13
RH4547 Mata cka2::kanMX4 his3 leu2 lys2 trp1 ura3 bar1 this study 
RH4551 Mata cka1::HIS3MX6 cka2::kanMX4 his3 leu2 lys2 trp1 ura3 bar1 pRS316::cka2-13::5MYC::6HIS this study 
RH4552 Mata cka1::HIS3MX6 cka2::kanMX4 his3 leu2 lys2 trp1 ura3 bar1 Ycplac22::cka1-13::3HA::6HIS this study 
RH4839 Mata HHF2::3HA::kanMX4 ade2 leu2 lys2 trp1 ura3 bar1 this study 
RH4841 Mata cka1::HIS3MX6 cka2::kanMX4 ade2 his3 leu2 lys2 trp1 ura3 bar1 Ycplac22::cka1-13::3HA::6HIS this study 
RH4845 Matα cka1::HIS3MX6 cka2::kanMX4 ade2 his3 leu2 lys2 trp1 ura3 bar1 pRS316::cka2-13::5MYC::6HIS this study 
RH4848 Mata cmd1-228 ade2 his3 leu2 lys2 trp1 ura3 bar1 this study 
RH4851 Mata cmd1-239 ade2 his3 leu2 lys2 trp1 ura3 bar1 this study 
RH4852 Mata cmd1-231 ade2 his3 leu2 lys2 trp1 ura3 bar1 this study 
DBY7443 Mata cmd1-231 his3 leu2 lys2 trp1 ura3 bar1 D. Botstein 
DBY7445 Mata cmd1-228 his3 leu2 lys2 trp1 ura3 bar1 D. Botstein 
DBY7446 Mata cmd1-226 his3 leu2 lys2 trp1 ura3 bar1 D. Botstein 
DBY7449 Mata cmd1-239 his3 leu2 lys2 trp1 ura3 bar1 D. Botstein 
ESM218 Mata tub4-1 ade2 his3 leu2 lys2 trp1 ura3 [48
RLY188 Mata arp3::HIS3 pRS316::ARP3::5MYC::6HIS his3 leu2 lys2 ura3 [7
YMW81 Mata arp2-1 ade2 his3 leu2 lys2 trp1 ura3 [5
Strain Relevant markers Reference 
RH2877 Mata ade2 leu2 lys2 trp1 ura3 bar1 Riezman collection 
RH2884 Matα ade2 his3 leu2 trp1 ura3 bar1 Riezman collection 
RH2887 Mata his3 leu2 lys2 trp1 ura3 bar1 Riezman collection 
RH3428 Mata arc35-1 ade2 leu2 trp1 ura3 bar1 this study 
RH3429 Mata arc35-1 ade2 his4 leu2 lys2 ura3 bar1 [13
RH3431 Mata/Matα arc35-1/ARC35 ade2/ADE2 his4/HIS4 leu2/leu2 lys2/LYS2 trp1/TRP1 ura3/ura3 bar1/bar1 [13
RH4547 Mata cka2::kanMX4 his3 leu2 lys2 trp1 ura3 bar1 this study 
RH4551 Mata cka1::HIS3MX6 cka2::kanMX4 his3 leu2 lys2 trp1 ura3 bar1 pRS316::cka2-13::5MYC::6HIS this study 
RH4552 Mata cka1::HIS3MX6 cka2::kanMX4 his3 leu2 lys2 trp1 ura3 bar1 Ycplac22::cka1-13::3HA::6HIS this study 
RH4839 Mata HHF2::3HA::kanMX4 ade2 leu2 lys2 trp1 ura3 bar1 this study 
RH4841 Mata cka1::HIS3MX6 cka2::kanMX4 ade2 his3 leu2 lys2 trp1 ura3 bar1 Ycplac22::cka1-13::3HA::6HIS this study 
RH4845 Matα cka1::HIS3MX6 cka2::kanMX4 ade2 his3 leu2 lys2 trp1 ura3 bar1 pRS316::cka2-13::5MYC::6HIS this study 
RH4848 Mata cmd1-228 ade2 his3 leu2 lys2 trp1 ura3 bar1 this study 
RH4851 Mata cmd1-239 ade2 his3 leu2 lys2 trp1 ura3 bar1 this study 
RH4852 Mata cmd1-231 ade2 his3 leu2 lys2 trp1 ura3 bar1 this study 
DBY7443 Mata cmd1-231 his3 leu2 lys2 trp1 ura3 bar1 D. Botstein 
DBY7445 Mata cmd1-228 his3 leu2 lys2 trp1 ura3 bar1 D. Botstein 
DBY7446 Mata cmd1-226 his3 leu2 lys2 trp1 ura3 bar1 D. Botstein 
DBY7449 Mata cmd1-239 his3 leu2 lys2 trp1 ura3 bar1 D. Botstein 
ESM218 Mata tub4-1 ade2 his3 leu2 lys2 trp1 ura3 [48
RLY188 Mata arp3::HIS3 pRS316::ARP3::5MYC::6HIS his3 leu2 lys2 ura3 [7
YMW81 Mata arp2-1 ade2 his3 leu2 lys2 trp1 ura3 [5

DNA manipulations and plasmid constructions

All DNA manipulations were performed according to standard techniques [34]. Restriction enzymes, Klenow, T4 DNA polymerase, polynucleotide kinase (PNK), calf intestine phosphatase (CIP), and T4 DNA ligase were obtained from Roche Diagnostics, New England Biolabs, United States Biochemical, Stratagene Cloning Systems, or MBI Fermentas. All DNA manipulations for cloning purposes were performed with the corresponding Qiagen kit. Transformation of E. coli was achieved by electroporation [35]. All polymerase chain reactions (PCRs) for cloning purposes were performed with a DNA polymerase with proof-reading activity (Vent, New England Biolabs). Oligonucleotides were synthesized by Microsynth GmbH (Balgach, Switzerland). All constructs were sequenced using a big dye terminator cycle sequencing ready reaction kit (Applied Biosystems Inc., Foster City, CA, USA).

All plasmids used in this study are listed in Table 2. All oligonucleotides for cloning purposes are listed in Table 3. YEplac181::ARC35 was constructed by removing the 3400-bp PstI/XbaI fragment from YCplac111::ARC35 followed by Klenow and T4 DNA polymerase treatment and ligation into YEplac181 [36], cut with SmaI and treated with CIP. The cmd1 mutant alleles for cloning into YEplac181 were amplified from genomic DNA of the strains DBY7446, DBY7445, DBY7449, or DBY7443 with the primers CMDASZ_3 and CMDASZ_4. The primers include a BamHI or SphI restriction site for cloning into YEplac181 [36]. For plasmid YEplac181::ARP3::5MYC::6HIS, the 1800-bp HindIII/SacI fragment from pDW20 [7] was ligated into YEplac181 [36], cut with HindIII and SacI and treated with CIP. Plasmid pRS316::5MYC::6HIS was constructed by cloning the 300-bp BamHI/EcoRI fragment from pDW20 [7] into pRS316 [37]. For construction of the high copy series plasmids of non-tagged CKII subunits, CKA1 was amplified with oligonucleotides CKA1_1 and CKA1_2 from genomic DNA of strain RH2877 and digested with HindIII and EcoRI, CKB1 with oligonucleotides CKB1_1 and CKB1_2 and digested with EcoRI and SalI, and CKB2 was amplified with oligonucleotides CKB2_1 and CKB2_2 and digested with EcoRI, followed by cloning into YEplac112 [36], pASZ12 [38], and YEplac181 [36], respectively. For 3HA-tagging, CKA1 was amplified with oligonucleotides CKA1_7 and CKA1_8, cut with EcoRV, and cloned into pFA::(GA)10::3HA::6HIS[39], cut open with SmaI, to produce pFA::CKA1::(GA)10::3HA::6HIS. To introduce the mutation D235N, which renders CKA1 catalytically inactive [40], PCR mutagenesis was performed on pFA::CKA1::(GA)10::3HA::6HIS with the oligonucleotides D352N_1 and D235N_2. The resulting PCR product was treated with DpnI to eliminate parental DNA and with PNK to phosphorylate the ends prior to religation to yield pFA::cka1-13::(GA)10::3HA::6HIS. For construction of the CEN- and -based cka1-13 vectors, cka1-13::(GA)10::3HA::6HIS was amplified from pFA::cka1-13::(GA)10::3HA::6HIS with oligonucleotides CKA1_9 and 6HISRV introducing a stop codon, cut with EcoRV, and cloned into YCplac22 [36], cut with SmaI to yield YCplac22::cka1-13::(GA)10::3HA::6HIS, and cloned into YEplac181 [36], cut with SmaI, to give YEplac181::cka1-13::(GA)10::3HA::6HIS. For MYC-tagging of CKA2 and CKB2, CKA2 was amplified with oligonucleotides CKA2_7 and CKA2_8 and CKB2 with oligonucleotides CKB2_7 and CKB2_8. Both PCR fragments were digested with BamHI and XbaI and cloned into pRS316::5MYC::6HIS, cut with BamHI and XbaI, to give plasmids pRS316::CKA2::5MYC::6HIS and pRS316::CKB2::5MYC::6HIS. For construction of YEplac181::CKA2::5MYC::6HIS and of YEplac181::CKB2::5MYC::6HIS, the XbaI/EcoRI fragments spanning CKA2::5MYC::6HIS or CKB2::5MYC::6HIS, respectively, were isolated from the corresponding pRS316::5MYC::6HIS plasmids and cloned into YEplac181 [36], cut with XbaI and EcoRI. The XbaI/EcoRI fragment from pRS316::CKA2::5MYC::6HIS was cloned into pBluescript, cut with XbaI and EcoRI, to give pBS::CKA2::5MYC::6HIS. PCR mutagenesis for production of the catalytically inactive cka2-13 allele carrying the mutation D225N [41] was performed as described for cka1-13 with the oligonucleotides D225N_1 and D225N_2 and pBS::CKA2::5MYC::6HIS as template. The resulting plasmid was pBS::cka2-13::5MYC::6HIS. To generate the CEN- and -based cka2-13::5MYC::6HIS-containing vectors, the XbaI/EcoRI fragment from pBS::cka2-13::5MYC::6HIS was cloned into pRS316 [37], cut with XbaI and EcoRI, to produce plasmid pRS316::cka2-13::5MYC::6HIS. YEplac181::cka2-13::5MYC::6HIS was constructed similarly using YEplac181 [36] as the vector part.

2

Plasmids

Plasmid Yeast ori Marker Insert Reference 
pBluescript (pBS) – – –  
pFA::3HA::6HIS – – (GA)10::3HA::6HIS [39
Ycplac22 CEN TRP1 – [36
pRS316 CEN URA3 – [37
pRS316::5MYC::6HIS CEN URA3 5MYC::6HIS this study 
Yeplac112  TRP1 – [36
Yeplac181  LEU2 – [36
Yeplac195  URA3 – [36
pASZ12  ADE2 – [38
Yeplac112::CKA1  TRP1 CKA1 this study 
pFA::CKA1::3HA::6HIS – – CKA1::(GA)10::3HA::6HIS this study 
pFA::cka1-13::3HA::6HIS – – cka1-13::(GA)10::3HA::6HIS this study 
Ycplac22::cka1-13::3HA::6HIS CEN TRP1 cka1-13::(GA)10::3HA::6HIS this study 
Yeplac181::cka1-13::3HA::6HIS  LEU2 cka1-13::(GA)10::3HA::6HIS this study 
pRS316::CKA2::5MYC::6HIS CEN URA3 CKA2::5MYC::6HIS this study 
Yeplac181::CKA2::5MYC::6HIS  LEU2 CKA2::5MYC::6HIS this study 
pBS::CKA2::5MYC::6HIS – – CKA2::5MYC::6HIS this study 
pBS::cka2-13::5MYC::6HIS – – cka2-13::5MYC::6HIS this study 
pRS316::cka2-13::5MYC::6HIS CEN URA3 cka2-13::5MYC::6HIS this study 
Yeplac181::cka2-13::5MYC::6HIS  LEU2 cka2-13::5MYC::6HIS this study 
pASZ12::CKB1  ADE2 CKB1 this study 
Yeplac181::CKB2  LEU2 CKB2 this study 
pRS316::CKB2::5MYC::6HIS CEN URA3 CKB2::5MYC::6HIS this study 
Yeplac181::CKB2::5MYC::6HIS  LEU2 CKB2::5MYC::6HIS this study 
pSD14  URA3 CMD1 S. Desrivières 
pASZ12::CMD1  ADE2 CMD1 [13
pASZ12::cmd1-226  ADE2 cmd1-226 [13
Yeplac181::cmd1-226  LEU2 cmd1-226 this study 
pASZ12::cmd1-228  ADE2 cmd1-228 [13
Yeplac181::cmd1-228  LEU2 cmd1-228 this study 
pASZ12::cmd1-239  ADE2 cmd1-239 [13
Yeplac181::cmd1-239  LEU2 cmd1-239 this study 
pASZ12::cmd1-231  ADE2 cmd1-231 [13
Yeplac181::cmd1-231  LEU2 cmd1-231 this study 
Ycplac111::ARC35 CEN LEU2 ARC35 Riezman collection 
Yeplac181::ARC35  LEU2 ARC35 this study 
pDW20 CEN URA3 ARP3::5MYC::6HIS [7
Yeplac181::ARP3::5MYC::6HIS  LEU2 ARP3::5MYC::6HIS this study 
pSM222 CEN URA3 TUB4::HA [48
pRS424::TUB4::3HA  TRP1 TUB4::3HA M. Snyder 
Plasmid Yeast ori Marker Insert Reference 
pBluescript (pBS) – – –  
pFA::3HA::6HIS – – (GA)10::3HA::6HIS [39
Ycplac22 CEN TRP1 – [36
pRS316 CEN URA3 – [37
pRS316::5MYC::6HIS CEN URA3 5MYC::6HIS this study 
Yeplac112  TRP1 – [36
Yeplac181  LEU2 – [36
Yeplac195  URA3 – [36
pASZ12  ADE2 – [38
Yeplac112::CKA1  TRP1 CKA1 this study 
pFA::CKA1::3HA::6HIS – – CKA1::(GA)10::3HA::6HIS this study 
pFA::cka1-13::3HA::6HIS – – cka1-13::(GA)10::3HA::6HIS this study 
Ycplac22::cka1-13::3HA::6HIS CEN TRP1 cka1-13::(GA)10::3HA::6HIS this study 
Yeplac181::cka1-13::3HA::6HIS  LEU2 cka1-13::(GA)10::3HA::6HIS this study 
pRS316::CKA2::5MYC::6HIS CEN URA3 CKA2::5MYC::6HIS this study 
Yeplac181::CKA2::5MYC::6HIS  LEU2 CKA2::5MYC::6HIS this study 
pBS::CKA2::5MYC::6HIS – – CKA2::5MYC::6HIS this study 
pBS::cka2-13::5MYC::6HIS – – cka2-13::5MYC::6HIS this study 
pRS316::cka2-13::5MYC::6HIS CEN URA3 cka2-13::5MYC::6HIS this study 
Yeplac181::cka2-13::5MYC::6HIS  LEU2 cka2-13::5MYC::6HIS this study 
pASZ12::CKB1  ADE2 CKB1 this study 
Yeplac181::CKB2  LEU2 CKB2 this study 
pRS316::CKB2::5MYC::6HIS CEN URA3 CKB2::5MYC::6HIS this study 
Yeplac181::CKB2::5MYC::6HIS  LEU2 CKB2::5MYC::6HIS this study 
pSD14  URA3 CMD1 S. Desrivières 
pASZ12::CMD1  ADE2 CMD1 [13
pASZ12::cmd1-226  ADE2 cmd1-226 [13
Yeplac181::cmd1-226  LEU2 cmd1-226 this study 
pASZ12::cmd1-228  ADE2 cmd1-228 [13
Yeplac181::cmd1-228  LEU2 cmd1-228 this study 
pASZ12::cmd1-239  ADE2 cmd1-239 [13
Yeplac181::cmd1-239  LEU2 cmd1-239 this study 
pASZ12::cmd1-231  ADE2 cmd1-231 [13
Yeplac181::cmd1-231  LEU2 cmd1-231 this study 
Ycplac111::ARC35 CEN LEU2 ARC35 Riezman collection 
Yeplac181::ARC35  LEU2 ARC35 this study 
pDW20 CEN URA3 ARP3::5MYC::6HIS [7
Yeplac181::ARP3::5MYC::6HIS  LEU2 ARP3::5MYC::6HIS this study 
pSM222 CEN URA3 TUB4::HA [48
pRS424::TUB4::3HA  TRP1 TUB4::3HA M. Snyder 
3

Oligonucleotides

name Sequence 
CMDASZ_3 5′-CGC GGA TCC ATG TAT TTA TAT TTT CGT GTA 
CMDASZ_4 5′-TGA CAT GCA TGC AGA ATG GTA AGG GTA AGA TAG 
CKA1_1 5′-CCC AAG CTT GAG GAC ATT GAG TCT CAG ATG AGA AAT CTA 
CKA1_2 5′-CCG GAA TTC CGT GTA TAA ATA CTC GAG CTG CAT CCT TTC 
CKA1_7 5′-AAA CTG CAG GAG GAC ATT GAG TCT CAG ATG AGA AAT CTA 
CKA1_8 5′-CCG GAT ATC TTT TTC AAT TTG TTC CCT TAT TGG GGC AAA 
D235N_1 5′-TTA AAC TTG TGG TCG TTT GGG ACA ATG TTG 
D235N_2 5′-AGA ATA ATC ATA CAT TCT GTA GTC AAC TAG 
CKA1_9 5′-CCG GAT ATC CTG CTG AGG AAG CGA ATA AAG ACG CAC AGG 
6HISRV 5′-CCG GAT ATC TTA TCA CTA ATG GTG ATG ATG GTG ATG AGC 
CKA2_7 5′-TGC TCT AGA CTC AAC TCT GAA GTT GAT TTA CTT GCT GTA 
CKA2_8 5′-CGC GGA TCC TTC AAA CTT CGT TTT GAA AAA CTT ATG ATC 
D225N_1 5′-CTA AAC TTA TGG TCA GTA GGA TGC ATG CTA 
D225N_2 5′-GGA GTA GTC ATA TTG GTT CAA GTT TAC TAA 
CKB1_1 5′-CCG GAA TTC CTT AGT CCA TTT GGC ATA ATT ATC CTG CGC 
CKB1_2 5′-CGC GTC GAC CAG TGA AGT TGA CCG TGG AGC TAA AGT ACT 
CKB2_1 5′-CCG GAA TTC CGC CAT CAC GTG ACC CCC ACT GCC TGC CAA 
CKB2_2 5′-CCG GAA TTC AAC CGC CTC TTA GTT CAT AGG CAG TCT CCA 
CKB2_7 5′-TGC TCT AGA CGC CAT CAC GTG ACC CCC ACT GCC TGC CAA 
CKB2_8 5′-CGC GGA TCC GGT TTT AAA ACC ACC ACT TTT CGT CAA ATC 
name Sequence 
CMDASZ_3 5′-CGC GGA TCC ATG TAT TTA TAT TTT CGT GTA 
CMDASZ_4 5′-TGA CAT GCA TGC AGA ATG GTA AGG GTA AGA TAG 
CKA1_1 5′-CCC AAG CTT GAG GAC ATT GAG TCT CAG ATG AGA AAT CTA 
CKA1_2 5′-CCG GAA TTC CGT GTA TAA ATA CTC GAG CTG CAT CCT TTC 
CKA1_7 5′-AAA CTG CAG GAG GAC ATT GAG TCT CAG ATG AGA AAT CTA 
CKA1_8 5′-CCG GAT ATC TTT TTC AAT TTG TTC CCT TAT TGG GGC AAA 
D235N_1 5′-TTA AAC TTG TGG TCG TTT GGG ACA ATG TTG 
D235N_2 5′-AGA ATA ATC ATA CAT TCT GTA GTC AAC TAG 
CKA1_9 5′-CCG GAT ATC CTG CTG AGG AAG CGA ATA AAG ACG CAC AGG 
6HISRV 5′-CCG GAT ATC TTA TCA CTA ATG GTG ATG ATG GTG ATG AGC 
CKA2_7 5′-TGC TCT AGA CTC AAC TCT GAA GTT GAT TTA CTT GCT GTA 
CKA2_8 5′-CGC GGA TCC TTC AAA CTT CGT TTT GAA AAA CTT ATG ATC 
D225N_1 5′-CTA AAC TTA TGG TCA GTA GGA TGC ATG CTA 
D225N_2 5′-GGA GTA GTC ATA TTG GTT CAA GTT TAC TAA 
CKB1_1 5′-CCG GAA TTC CTT AGT CCA TTT GGC ATA ATT ATC CTG CGC 
CKB1_2 5′-CGC GTC GAC CAG TGA AGT TGA CCG TGG AGC TAA AGT ACT 
CKB2_1 5′-CCG GAA TTC CGC CAT CAC GTG ACC CCC ACT GCC TGC CAA 
CKB2_2 5′-CCG GAA TTC AAC CGC CTC TTA GTT CAT AGG CAG TCT CCA 
CKB2_7 5′-TGC TCT AGA CGC CAT CAC GTG ACC CCC ACT GCC TGC CAA 
CKB2_8 5′-CGC GGA TCC GGT TTT AAA ACC ACC ACT TTT CGT CAA ATC 

α-Factor uptake

35S α-factor uptakes were performed as described [42]. The average values from three independent experiments were calculated and plotted. The standard deviation is indicated.

Immunofluorescence

Indirect immunofluorescence for staining of β-tubulin was performed as described [14].

To visualize nuclear DNA, 108 cells were resuspended in 1 ml 96% ethanol and incubated for 5 min at room temperature (RT). DAPI (4,6-diamidino-2-phenylindole) was added to a final concentration of 1 μg ml−1 followed by a 1-min incubation. The cells were then washed three times in sterile water and resuspended in 100 μl 50% glycerol.

Immunofluorescence was observed with a Zeiss Axiophot microscope equipped with a 100× objective. Images were taken with a Zeiss camera coupled to a video system (MWG Biotech). Exposure time and other processing parameters were adjusted to the signal strength of the sample.

Western blotting and antibodies

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was used for detection of Cmd1p, Hhf2p::3HA, Arc35p, Cka2p::5MYC, Ckb2p::5MYC, Hxk1p, Mcm1p, Arp3::5MYC, Tub4p::3HA, and Spc110p. Western blotting was performed as described [13]. The antibodies against Cmd1p and Arc35p have been described [13,43] and were used at a dilution of 1/1000. Hxk1p was detected with a polyclonal antibody (M. Hall, Basel, Switzerland) used at a dilution of 1/1000. The antibody against Mcm1p (M. Hall), the 9E10 antibody (Novartis, Basel, Switzerland) for detection of the 5MYC-tagged proteins, and the mouse 12CA5 antibody (Roche Diagnostics) for detection of 3HA-tagged proteins were used at a dilution of 1/800.

Separation of nuclei from cytosol

To derive nuclear and cytosolic fractions a previous protocol [44] was modified. 1010 cells were collected and resuspended in 50 ml spheroplasting buffer (1.2 M sorbitol, 30 mM 2-mercaptoethanol, 5000 U recombinant lyticase in 20 mM KPi, pH 7.4) for 90 min at 24°C. The spheroplasts were washed once in 50 ml 1.2 M sorbitol in 20 mM KPi, pH 7.4, resuspended in 10 ml lysis buffer (18% (w/v) Ficoll, 0.5 mM MgCl2, 1 μg ml−1 each leupeptin, pepstatin, antipain, chymostatin, aprotinin in 20 mM Kpi, pH 6.4), and lysed by gently inverting the tube. The homogenate was diluted 1:1 with dilution buffer (2.4 M sorbitol, 0.5 mM MgCl2, 1 μg ml−1 each leupeptin, pepstatin, antipain, chymostatin, aprotinin in 20 mM Kpi, pH 6.4) and spun for 10 min at 2500 rpm in a Sorvall HB-4 rotor. The supernatant was transferred into a fresh tube, underlaid with 1 ml cushion buffer (2 M sucrose, 0.5 mM MgCl2, 1 μg ml−1 each leupeptin, pepstatin, antipain, chymostatin, aprotinin in 20 mM Kpi, pH 6.4), and spun for 30 min at 12 000 rpm in a Sorvall HB-4 rotor. The majority of the supernatant was removed as cytosol, the interphase and cushion were pooled as nuclei, and each fraction was brought to 20 ml with a 1:1 mixture of lysis buffer/dilution buffer. The two fractions were underlaid with 1 ml cushion buffer each and spun for 30 min at 12 000 rpm in a Sorvall HB-4 rotor; 1 ml of the supernatant from the cytosol sample was kept as cytosol. From the nuclear sample, the interphase and cushion were collected, diluted to 6 ml with lysis buffer, mixed by gentle inversion, and loaded onto a sucrose gradient containing steps of 1.9 M (5 ml), 1.4 M (10 ml), and 1.2 M (5 ml) sucrose in 9% (w/v) Ficoll, 0.5 mM MgCl2, 1 μg ml−1 each leupeptin, pepstatin, antipain, chymostatin, aprotinin, 20 mM Kpi, pH 6.4. Following a centrifugation of 60 min at 25 000 rpm in a Kontron TST 28.38 rotor, the 1.2/1.4 M and 1.4/1.9 M interphases were collected and pooled as nuclei. 100% trichloroacetic acid was added to a final concentration of 10% to the cytosol and nuclei samples. The samples were incubated on ice for 30 min, proteins were collected by centrifugation, washed once in acetone, and resuspended in 300 μl 125 mM Tris, pH 6.8, 2% SDS, 0.1 M dithiothreitol (DTT), 30% (v/v) glycerol, 5% (v/v) β-mercaptoethanol (2×fetal scalp blood (FSB)). The samples were boiled for 15 min while shaking and centrifuged for 5 min; 35 μl of each fraction were analyzed by SDS–PAGE and immunoblotting.

Co-immunoprecipitations from cytosol

Cytosol was prepared from 5×109 cells as described above except for the addition of 5 mM CaCl2 to all buffers. The recovered cytosol was diluted 1:1 with IP buffer (20 mM KPi, pH 6.4, 0.5 mM MgCl2, 5 mM CaCl2, 1 μg ml−1 each leupeptin, pepstatin, antipain, chymostatin, aprotinin) and split in two. 80 μl 50% protein A-Sepharose beads were added to both samples; 60 μl α-Cmd1p antibody was added to one of the two reactions. The samples were incubated for 2 h at 4°C while shaking. Following a brief centrifugation, supernatant and pellet were separated. 1 ml of the supernatant was TCA precipitated and resuspended in 150 μl 2×FSB. The pellet from the immunoprecipitation was taken up in 1 ml of a 1:1:2 mixture of lysis buffer/dilution buffer/IP buffer, subjected to three washes, and resuspended in 600 μl 2×FSB. Both supernatant and pellet were boiled for 5 min and centrifuged for 5 min; 20 μl of each fraction were analyzed by SDS–PAGE and immunoblotting.

Glycerol gradients

2×109 cells were collected by centrifugation, resuspended in 100 ml 0.1 M Tris–SO4, pH 9.4, 28 mM 2-mercaptoethanol, and incubated at RT for 20 min. The collected cells were resuspended in 20 ml 2% yeast extract, 1% peptone, 0.7 M sorbitol, 10 mM Tris, pH 8.0, 3500 U recombinant lyticase, incubated at RT with gentle shaking for 45 min, collected, washed in 20 ml 0.7 M sorbitol, 20 mM Tris, pH 8.0, 2 mM ethylenediamine tetraacetic acid (EDTA), 0.1 mM DTT, resuspended in 1 ml TNED (20 mM Tris, pH 8.0, 2 mM EDTA, 0.1 mM DTT, 1% (v/v) NP-40), and lysed by 40 strokes in a dounce homogenizer on ice. The lysate was cleared by a low-speed centrifugation; 500 μg of protein in a total volume of 150 μl were loaded onto the glycerol gradient. A 5-ml step gradient from 15% (v/v) to 40% (v/v) glycerol in TNED was prepared and centrifuged at 48 680 rpm in a TST55.5 rotor (Centrikon) for 5 h at 4°C. 270-μl Fractions were collected from the top, precipitated with trichloroacetic acid, resuspended in 100 μl 2×FSB, boiled with shaking for 15 min and centrifuged for 5 min. 20 μl of each fraction were analyzed by immunoblotting for Arp3::5MYC and Arc35p.

Results

Overexpression of CKII subunits suppress arc35-1 temperature sensitivity

ARC35 has been identified in a screen for endocytosis-deficient mutants [45] and biochemically as a component of the Arp2/3 complex [7]. Analysis of the arc35-1 ts strain suggested that Arc35p regulates two cellular functions: endocytosis via organization of the actin cytoskeleton [13] and cell cycle progression by affecting the tubulin cytoskeleton [14]. Genetic data indicated that the two functions of Arc35p are mediated through distinct calmodulin pathways [13,14].

In order to understand better the functions of Arc35p, dosage-dependent suppressors of the temperature sensitivity of arc35-1 were isolated. One candidate clone encoding CKB2, the β′ regulatory subunit of yeast CKII [27], restored growth of arc35-1 at 37°C (Fig. 1A) without affecting growth of the isogenic wild-type (Wt) strain (data not shown). We next determined if the three other subunits of yeast CKII, CKA1, CKA2, and CKB1, could also suppress arc35-1 temperature sensitivity upon overexpression. As for Ckb2p, all three proteins promoted growth at 37°C when compared to the empty control plasmid (Fig. 1A). Suppression of arc35-1 by CKII required catalytic activity of the kinase because kinase-dead alleles of both CKA1 (cka1-13, [40]) and CKA2 (cka2-13, [41]) failed to suppress ts growth upon overexpression (Fig. 1A). We next determined whether temperature sensitivity of mutants defective in other subunits of the Arp2/3 complex could be suppressed by CKII. Overexpression of either CKA1, CKA2, CKB1, or CKB2 in an arp2-1 background (Fig. 1A, III, a–d) or an arp3-2 background (data not shown) did not restore growth at 37°C, similar to what has been observed previously for overexpression of Cmd1p in the identical mutant strains [13]. Genetic interaction of CKII with the Arp2/3 complex may therefore be limited to Arc35p.

1

Overexpression of Ckb2p restores the integrity of the tubulin cytoskeleton, but not endocytosis, in the arc35-1 mutant at 37°C. A: arc35-1 (RH3428 and RH3429) (I and II) and arp2-1 (YMW81) (III and IV) mutant strains were transformed with the following plasmids: YEplac112::CKA1 (a), YEeplac181::CKA2::5MYC::6HIS (b), pASZ12::CKB1 (c), YEeplac181::CKB2::5MYC::6HIS (d), YEplac181::cka1-13::3HA::6HIS (e), YEplac181::cka2-13::5MYC::6HIS (f), pASZ12::CMD1 (g), pRS424::TUB4::3HA (h), pASZ12 (i), YEplac112 (j), YEplac181::ARC35 (k). The strains were grown on YPUADT for 2 days at 37°C. B: An arc35-1 strain (RH3429) bearing plasmid YEplac181 (a) or plasmid YEplac181::CKB2 (b) was grown in selective medium at 24°C, preincubated at 37°C for 3 h, fixed, and processed for tubulin staining. Cells were visualized by epifluorescence. The size bar corresponds to 5 μm. C: A Wt strain (RH2877) and an arc35-1 strain (RH3429) without plasmid or bearing plasmid YEplac181::CKB2 were preincubated for 20 min at 24 or 37°C and assayed for their ability to internalize radioactive α-factor. The results are presented as the percentage of radioactive α-factor internalized (y-axis) for each time point (x-axis).

1

Overexpression of Ckb2p restores the integrity of the tubulin cytoskeleton, but not endocytosis, in the arc35-1 mutant at 37°C. A: arc35-1 (RH3428 and RH3429) (I and II) and arp2-1 (YMW81) (III and IV) mutant strains were transformed with the following plasmids: YEplac112::CKA1 (a), YEeplac181::CKA2::5MYC::6HIS (b), pASZ12::CKB1 (c), YEeplac181::CKB2::5MYC::6HIS (d), YEplac181::cka1-13::3HA::6HIS (e), YEplac181::cka2-13::5MYC::6HIS (f), pASZ12::CMD1 (g), pRS424::TUB4::3HA (h), pASZ12 (i), YEplac112 (j), YEplac181::ARC35 (k). The strains were grown on YPUADT for 2 days at 37°C. B: An arc35-1 strain (RH3429) bearing plasmid YEplac181 (a) or plasmid YEplac181::CKB2 (b) was grown in selective medium at 24°C, preincubated at 37°C for 3 h, fixed, and processed for tubulin staining. Cells were visualized by epifluorescence. The size bar corresponds to 5 μm. C: A Wt strain (RH2877) and an arc35-1 strain (RH3429) without plasmid or bearing plasmid YEplac181::CKB2 were preincubated for 20 min at 24 or 37°C and assayed for their ability to internalize radioactive α-factor. The results are presented as the percentage of radioactive α-factor internalized (y-axis) for each time point (x-axis).

Overexpression of Ckb2p restores the integrity of the tubulin cytoskeleton, but neither endocytosis nor the integrity of the actin cytoskeleton, in the arc35-1 mutant at 37°C

Since the ts growth of arc35-1 could be caused either by the actin/endocytosis defect and/or by the tubulin defect, we wanted to determine if the two defects were suppressed by overexpression of Ckb2p. An arc35-1 strain bearing the empty control plasmid or expressing Ckb2p was preincubated at 37°C for 3 h, fixed, and processed for tubulin immunolocalization (Fig. 1B). The arc35-1 strain displayed defects in the organization of the tubulin cytoskeleton, seen as an absence of anaphase spindles [14]. The strain accumulated large budded cells with a short spindle positioned adjacent to the bud neck indicative of metaphase spindles. The same strain overexpressing Ckb2p exhibited Wt organization of the tubulin cytoskeleton as described [46]. The presence of elongated anaphase spindles was obvious.

To assay for suppression of the endocytic defect of arc35-1 at 37°C, an arc35-1 strain without plasmid or overexpressing Ckb2p was assayed for its ability to internalize radioactive α-factor (Fig. 1C). The mutant strain without plasmid (arc35-1) showed reduced kinetics of internalization at 24°C when compared to a Wt strain and virtually no internalization at 37°C as described [45]. Overexpression of Ckb2p (arc35-1+CKB2) did not restore the uptake kinetics at 37°C. None of the other three subunits could restore endocytosis in arc35-1 upon overexpression (data not shown). Overexpression of the CKII subunits in the Wt background did not affect endocytosis (data not shown). In agreement with previous findings [13], the defect in organization of the actin cytoskeleton of the arc35-1 mutant as judged by phalloidin staining could not be suppressed by overexpression of CKII subunits, emphasizing a tight linkage of the actin and endocytosis defects in the arc35-1 strain. CKII therefore suppresses defects of the arc35-1 mutant in the tubulin cytoskeleton, but not in endocytosis.

ARC35, CKII, CMD1, and TUB4 interact genetically

We next tested whether overexpression of other genes leading to metaphase arrest upon mutation could suppress the temperature sensitivity of the arc35-1 mutant. Only the yeast γ-tubulin Tub4p was found to do so (Fig. 1A). Interestingly, there is a striking correlation of mutant phenotypes among certain mutants in ARC35, CKII, CMD1, and TUB4. First, the arc35-1 mutant, besides a defect in the organization of the actin cytoskeleton, undergoes metaphase arrest upon shift to the restrictive temperature [14]. This phenotype is not found upon mutation of other subunits of the Arp2/3 complex such as Arp2 or Arp3 (unpublished observation). Second, alleles of CKA2 such as cka2-13, which are ts for both kinase activity and growth, are defective for passage from G1 to S phase and for initiation/completion of mitosis arresting with metaphase and anaphase spindles [41]. The identical mutation in CKA1, cka1-13, leads to growth arrest without any obvious cell cycle defect [40]. Third, mutations in CMD1 can be divided into at least four distinct classes [47]. One class of mutants represented by the cmd1-239 allele undergoes metaphase arrest. The other, non-mitotic alleles show defects in organization of the actin cytoskeleton (cmd1-226), in localization of Cmd1p (cmd1-228), or in bud emergence (cmd1-231). Fourth, certain alleles of the yeast γ-tubulin TUB4 (tub4-1) undergo complete metaphase arrest due to alterations in the spindle structure [48,49].

In order to gain better insight into the genetic interactions between ARC35, CKII, CMD1, and TUB4, the corresponding genes were overexpressed in arc35, arp2, cka1, cka2, cmd1, and tub4 mutant strains, and growth of the resulting strains was monitored at 37°C (Fig. 1A and Table 4). Overexpression of Arc35p could suppress only the ts growth of arc35-1, but of no other mutant. Overexpression of CKII subunits could only suppress ts growth of the arc35-1 strain as described above. Overexpression of CMD1 was found to suppress ts growth of the metaphase-arresting strains arc35-1 and cka2-13. Temperature sensitivity of the non-mitotic arp2-1 and cka1-13 alleles could not be overcome by overexpression of Cmd1p. Ts growth of the metaphase-arresting cka2-13 strain was overcome by overexpression of all cmd1 mutant alleles that do not display a mitotic defect. Overexpression of the metaphase-arresting cmd1-239 allele did not restore growth to the cka2-13 mutant. Consistent with this, overexpression of Cmd1-239p in an arc35-1 background did not suppress ts growth [14]. Furthermore, growth at 37°C could be restored to the metaphase-arresting arc35-1, cka2-13, and cmd1-239 strains upon overexpression of TUB4. Temperature sensitivity of strains expressing either arp2-1 or other alleles of the corresponding genes, which do not undergo metaphase arrest, could not be suppressed by high levels of Tub4p. None of the mutations was found to be dominant. Suppression of ts growth correlated with appearance of anaphase spindles following shift to 37°C in all cases (Fig. 2). In support of the observed increase in anaphase spindle formation, the percentage of cells displaying a metaphase spindle decreased to almost Wt numbers for the mutants at 37°C when their growth phenotype was rescued by overexpression of the indicated gene products.

4

Genetic interactions between the Arp2/3 complex, CKII, CMD1, and TUB4

2mu;:: Strain 
 arc35-1 arp2-1 cka2-13 cka1-13 cmd1-239 cmd1-228 tub4-1 
ARC35 − − − − − − 
ARP3 − − − − − − − 
CKA1 − n.d. − − − 
CKA2 − − − − 
CKB1 − − − − − 
CKB2 − − − − − 
cka1-13 − n.d. n.d. n.d. n.d. n.d. n.d. 
cka2-13 − n.d. n.d. n.d. n.d. n.d. n.d. 
CMD1 − − − 
cmd1-226 n.d. n.d. n.d. 
cmd1-228 − n.d. n.d. − n.d. 
cmd1-239 − n.d. − n.d. − n.d. 
cmd1-231 n.d. n.d. n.d. 
TUB4 − − − 
2mu;:: Strain 
 arc35-1 arp2-1 cka2-13 cka1-13 cmd1-239 cmd1-228 tub4-1 
ARC35 − − − − − − 
ARP3 − − − − − − − 
CKA1 − n.d. − − − 
CKA2 − − − − 
CKB1 − − − − − 
CKB2 − − − − − 
cka1-13 − n.d. n.d. n.d. n.d. n.d. n.d. 
cka2-13 − n.d. n.d. n.d. n.d. n.d. n.d. 
CMD1 − − − 
cmd1-226 n.d. n.d. n.d. 
cmd1-228 − n.d. n.d. − n.d. 
cmd1-239 − n.d. − n.d. − n.d. 
cmd1-231 n.d. n.d. n.d. 
TUB4 − − − 

Mutant strains cka2-13 (RH4845), cka1-13 (RH4841), cmd1-239 (RH4851), cmd1-228 (RH4848), and tub4-1 (ESM218) were transformed with the following plasmids as indicated: YEplac181::ARC35, YEplac181::ARP3::5MYC::6HIS, YEplac112::CKA1, YEplac181::CKA2::5MYC::6HIS, pASZ12::CKB1, YEplac181::CKB2::5MYC::6HIS, YEplac181::cka1-13::3HA::6HIS, YEplac181::cka2-13::5MYC::6HIS, pSD14, YEplac181::cmd1-226, YEplac181::cmd1-228, YEplac181::cmd1-239, YEplac181::cmd1-231 (pASZ12::CMD1/::cmd1-226/::cmd1-228/::cmd1-239/::cmd1-231 for RH4845, respectively), and pRS424::TUB4::3HA. The strains were grown on YPUADT plates for 2 days at 37°C and growth was determined.

2

Suppression of ts growth correlates with the appearance of anaphase spindles in arc35-1, cka2-13, and cmd1-239. Wt (RH2877), arc35-1 (RH3429), cka2-13 (RH4845), and cmd1-239 (RH4851) strains were transformed with YEplac181::ARC35, YEplac181::CKA2::5MYC::6HIS, pASZ12::CMD1, or pRS424::TUB4::3HA as indicated. The resulting strains were grown at 24°C, preincubated at 37°C for 4 h, fixed, and processed for DAPI staining of the nuclear DNA. The percentage of cells with elongated anaphase spindles and short metaphase spindles positioned at the bud neck in the population (n=250) was determined and compared to the Wt control.

2

Suppression of ts growth correlates with the appearance of anaphase spindles in arc35-1, cka2-13, and cmd1-239. Wt (RH2877), arc35-1 (RH3429), cka2-13 (RH4845), and cmd1-239 (RH4851) strains were transformed with YEplac181::ARC35, YEplac181::CKA2::5MYC::6HIS, pASZ12::CMD1, or pRS424::TUB4::3HA as indicated. The resulting strains were grown at 24°C, preincubated at 37°C for 4 h, fixed, and processed for DAPI staining of the nuclear DNA. The percentage of cells with elongated anaphase spindles and short metaphase spindles positioned at the bud neck in the population (n=250) was determined and compared to the Wt control.

In summary, the arc35-1 allele was suppressed by overexpression of CKII, CMD1, and TUB4. The cka2-13 allele was suppressed by CMD1 and TUB4. The cmd1-239 allele was suppressed by TUB4. Ts growth of tub4-1 could not be rescued by overexpression of any gene analyzed. The genetic data presented provide evidence for an involvement of Arc35p, CKII, Cmd1p, and Tub4p in the same process.

Subcellular fractionation of Arc35p, CKII, Cmd1p, and Tub4p

Since a nuclear as well as cytosolic localization has been demonstrated for Cmd1p [50,51] and for Tub4p [48,52], it was of interest to determine whether Arc35p, CKII, Cmd1p, and Tub4p could possibly interact functionally in the cytosol or in the nucleus. Even though Arc35p has been localized to the cell cortex [13], a nuclear pool cannot be excluded. No localization data concerning CKII are available for yeast.

We determined the subcellular localization of the involved proteins using biochemical methods. For this purpose, a Wt strain expressing 3HA-tagged histone 4 (Hhf2p::3HA) without plasmid or bearing plasmid pDW20 (Arp3::5MYC::6HIS), pRS316::CKA2::5MYC::6HIS, pRS316::CKB2::5MYC::6HIS, or pSM222 (Tub4p::HA) was used for isolation of enriched nuclei and cytosol. Distribution of Arc35p, Arp3, Cka2p, Ckb2p, Cmd1p, and Tub4p was determined by immunoblotting. The data were corrected for cross-contamination of nuclear and cytosolic fractions using marker proteins, hexokinase for the cytosolic fraction, histone H4 and Mcm1p for the nuclear fraction (Fig. 3A). Arc35p was recovered almost exclusively in the cytosolic fraction (Fig. 3A and B). A majority of both Arp3 and Cmd1p were detected in the cytosol (Fig. 3B). The CKII subunits Cka2p and Ckb2p as well as Tub4p were found predominantly in the cytosol even though there was a clear nuclear pool (Fig. 3B). These data correlate with findings using green fluorescent protein (GFP) fusions (data not shown).

3

Subcellular fractionation of Arc35p, CKII, Cmd1p, Tub4p, and Spc110p. A: Cytosol and enriched nuclei were isolated from a Wt strain expressing Hhf2p::3HA (RH4839) grown at 30°C. The distribution of Arc35p was determined by immunoblotting and compared to the control proteins Hxk1p, Hhf2p::3HA, and Mcm1p. B: A Wt strain expressing Hhf2p::3HA (RH4839) grown at 30°C was used for isolation of cytosol and enriched nuclei. The relative distribution of Arc35p and Cmd1p as percentage of the recovered protein in either the nuclear or cytosolic fraction was determined by immunoblotting and densiometry. The numbers were corrected for contamination of cytosol by nuclei and of nuclei by cytosol based on Hxk1p and Hhf2p::3HA immunoblots. Cytosol and enriched nuclei were isolated from the identical strain (RH4839) bearing plasmid pDW20, pRS316::CKA2::5MYC::6HIS, pRS316::CKB2::5MYC::6HIS, or pSM222, and the relative distribution of Arp3::5MYC, Cka2p::5MYC, Ckb2p::5MYC, or Tub4p::HA, respectively, determined by immunoblotting as described above.

3

Subcellular fractionation of Arc35p, CKII, Cmd1p, Tub4p, and Spc110p. A: Cytosol and enriched nuclei were isolated from a Wt strain expressing Hhf2p::3HA (RH4839) grown at 30°C. The distribution of Arc35p was determined by immunoblotting and compared to the control proteins Hxk1p, Hhf2p::3HA, and Mcm1p. B: A Wt strain expressing Hhf2p::3HA (RH4839) grown at 30°C was used for isolation of cytosol and enriched nuclei. The relative distribution of Arc35p and Cmd1p as percentage of the recovered protein in either the nuclear or cytosolic fraction was determined by immunoblotting and densiometry. The numbers were corrected for contamination of cytosol by nuclei and of nuclei by cytosol based on Hxk1p and Hhf2p::3HA immunoblots. Cytosol and enriched nuclei were isolated from the identical strain (RH4839) bearing plasmid pDW20, pRS316::CKA2::5MYC::6HIS, pRS316::CKB2::5MYC::6HIS, or pSM222, and the relative distribution of Arp3::5MYC, Cka2p::5MYC, Ckb2p::5MYC, or Tub4p::HA, respectively, determined by immunoblotting as described above.

The localization data favor a model in which the four proteins work together in the cytosol even though a cascade from the cytosol to the nucleus cannot be excluded.

The Arp2/3 complex, CKII, Cmd1p, and Tub4p interact in the cytosol

A genetic interaction has been demonstrated for ARC35, CKII, CMD1, and TUB4. It was therefore of interest to determine whether those proteins interact biochemically. For this purpose, Cmd1p was immunoprecipitated from cytosol isolated from Wt strains expressing Arp3::5MYC::6HIS, Cka2p::5MYC::6HIS, Ckb2p::5MYC::6HIS, or Tub4p::HA. Calcium was included in the incubation because the interaction of Arc35p with Cmd1p was shown to depend on the presence of calcium [13]. The presence of Arc35p, Arp3p, Cka2p, Ckb2p, and Tub4p in the immunoprecipitate was then assayed by immunoblotting (Fig. 4A). Both Arc35p and Arp3p co-immunoprecipitated partially with Cmd1p. The specificity of this co-immunoprecipitation has been demonstrated previously [13]. In addition, some Cka2p, Ckb2p, and Tub4p were found to co-precipitate with Cmd1p. None of the proteins was recovered in the pellet in the absence of antibody (prot. A).

4

The Arp2/3 complex, CKII, Cmd1p, and Tub4p interact in the cytosol. A: A Wt strain (RH4839) grown at 24°C expressing either Arp3::5MYC (from pDW20), Cka2p::5MYC (from pRS316::CKA2::5MYC::6HIS), Ckb2p::5MYC (from pRS316::CKB2::5MYC::6HIS), or Tub4p::3HA (from pSM222) was used for isolation of cytosol. Protein A-Sepharose (prot. A) or protein A-Sepharose and α-Cmd1p serum (α-Cmd1p) was used for immunoprecipitation. Immunoprecipitates (P) and supernatants (S) were immunoblotted for Cmd1p, Arc35p, Arp3::5MYC, Cka2p::5MYC, Ckb2p::5MYC, or Tub4p::3HA, respectively. B: Cytosol was prepared from cmd1-239 (RH4851) and cmd1-231 (RH4852) mutant strains expressing Cka2p::5MYC (from pRS316::CKA2::5MYC::6HIS), Ckb2p::5MYC (from pRS316::CKB2::5MYC::6HIS), or Tub4p::3HA (from pSM222) grown at 24°C. Protein A-Sepharose and α-Cmd1p serum (α-Cmd1p) were used for immunoprecipitation. Immunoprecipitates (P) and supernatants (S) were immunoblotted for Cmd1p, Cka2p::5MYC, Ckb2p::5MYC, or Tub4p::3HA, respectively.

4

The Arp2/3 complex, CKII, Cmd1p, and Tub4p interact in the cytosol. A: A Wt strain (RH4839) grown at 24°C expressing either Arp3::5MYC (from pDW20), Cka2p::5MYC (from pRS316::CKA2::5MYC::6HIS), Ckb2p::5MYC (from pRS316::CKB2::5MYC::6HIS), or Tub4p::3HA (from pSM222) was used for isolation of cytosol. Protein A-Sepharose (prot. A) or protein A-Sepharose and α-Cmd1p serum (α-Cmd1p) was used for immunoprecipitation. Immunoprecipitates (P) and supernatants (S) were immunoblotted for Cmd1p, Arc35p, Arp3::5MYC, Cka2p::5MYC, Ckb2p::5MYC, or Tub4p::3HA, respectively. B: Cytosol was prepared from cmd1-239 (RH4851) and cmd1-231 (RH4852) mutant strains expressing Cka2p::5MYC (from pRS316::CKA2::5MYC::6HIS), Ckb2p::5MYC (from pRS316::CKB2::5MYC::6HIS), or Tub4p::3HA (from pSM222) grown at 24°C. Protein A-Sepharose and α-Cmd1p serum (α-Cmd1p) were used for immunoprecipitation. Immunoprecipitates (P) and supernatants (S) were immunoblotted for Cmd1p, Cka2p::5MYC, Ckb2p::5MYC, or Tub4p::3HA, respectively.

Genetic data suggest that the functional interaction of these proteins with Cmd1p is defective in the cmd1-239 mutant [13,14] (this publication). In addition, Cmd1-239p fails to interact with Arc35p [13]. It is therefore possible that the novel interactions demonstrated here would be affected by mutation of Cmd1p at this critical site. Therefore, cytosol was prepared from cmd1-239 and cmd1-231 mutant strains expressing tagged versions of Cka2p, Ckb2p, and Tub4p from plasmids and used for immunoprecipitation of Cmd1p as described. Immunoblotting showed that Cka2p and Ckb2p failed to co-precipitate with Cmd1-239p. The association of Tub4p with Cmd1-239p was less strongly affected (Fig. 4B). Both CKII subunits and Tub4p interacted with Cmd1-231p as well as with Wt Cmd1p. Lack of interaction of CKII subunits with Cmd1-239p, but not with Cmd1-231p, strongly supports the specificity of the observed interaction.

In summary, the data demonstrate biochemical interactions between the Arp2/3 complex, CKII subunits, Cmd1p, and Tub4p. These interactions depend on integrity of the calmodulin site defective in Cmd1-239p.

Arc35p and Arp3 cofractionate on a glycerol gradient

The arc35-1 mutant was found to have defects in the organization of the actin and tubulin cytoskeletons. The latter can be suppressed by overexpression of Cmd1p, CKII, and Tub4p [14] (this publication). The genetic interaction observed between ARC35, CMD1, CKII, and TUB4 seems to be specific for the Arc35 subunit of the Arp2/3 complex as it is not observed for the other subunits tested. This raises the possibility that Arc35p could exist in two distinct cellular complexes, in the Arp2/3 complex and in another complex with Cmd1p, CKII, and Tub4p. Therefore, we determined whether all Arc35p fractionates together with the Arp2/3 complex. Soluble protein extracts were prepared from a Wt strain expressing Arp3::5MYC::6HIS, and 500 μg of total protein were separated on a 15–40% glycerol gradient. The Arp3 content in each fraction was determined by immunoblotting (Fig. 5A). Arp3 was found in at least two peaks: one Arp3-containing complex (fractions 5 and 6) cofractionated with catalase at a molecular mass of 240 kDa corresponding to the previously seen molecular mass of the Arp2/3 complex [7]. Arp3 was identified in a second complex (9–11) with a molecular mass of approximately 500 kDa. The appearance of this high molecular mass complex may be explained with the interaction of the Arp2/3 complex with type I myosins, Vrp1p, and Las17p [53,54].

5

Arc35p and Arp3 cofractionate on a glycerol gradient. A: 500 μg of total protein extracts from a strain expressing Arp3::5MYC (RLY188) were separated on a glycerol gradient. The fractions collected were immunoblotted for Arp3::5MYC and Arc35p. The marker proteins aldolase (160 kDa), catalase (240 kDa), ferritin (440 kDa), and thyroglobulin (660 kDa) were identified in fractions 3, 5, 8, and 11, respectively. B: The percentage of total Arp3 and total Arc35p in each fraction was determined from the immunoblots in A and plotted against the fraction number.

5

Arc35p and Arp3 cofractionate on a glycerol gradient. A: 500 μg of total protein extracts from a strain expressing Arp3::5MYC (RLY188) were separated on a glycerol gradient. The fractions collected were immunoblotted for Arp3::5MYC and Arc35p. The marker proteins aldolase (160 kDa), catalase (240 kDa), ferritin (440 kDa), and thyroglobulin (660 kDa) were identified in fractions 3, 5, 8, and 11, respectively. B: The percentage of total Arp3 and total Arc35p in each fraction was determined from the immunoblots in A and plotted against the fraction number.

The same gradient fractions were used for detection of Arc35p by immunoblotting. The fractionation pattern of Arc35p was the same as for Arp3 (Fig. 5A and B). No fraction was found that contained Arc35p, but not Arp3. Therefore, it is most likely that all Arp35p is associated with the Arp2/3 complex. Still, the existence of a very minor amount of a second, Arc35p-containing complex cannot be excluded.

Discussion

In this study, we provide biochemical and genetic evidence for a role of Arc35p in control of the tubulin cytoskeleton via calmodulin, CKII, and γ-tubulin. These findings provide a basis to investigate the mechanism of how the Arp2/3 complex, thought to be mainly involved in actin dynamics, can also interact functionally with the tubulin cytoskeleton. The biochemical studies provide direct evidence for a physical interaction between Arc35p and elements known to affect the tubulin network, CKII and Tub4p. The genetic studies provide evidence that these proteins act in the same pathway.

Originally we found that the gene for the β′ regulatory subunit of yeast CKII, CKB2, was active as a dosage-dependent suppressor of the cell cycle, but not endocytic, defect of the arc35-1 mutant. However, each other subunit of CKII can also suppress arc35-1 ts mutant. This suggests that overproduction of any of the subunits of this enzyme can lead to increased CKII activity. Consistent with this hypothesis, kinase-dead alleles of both CKA1 and CKA2 failed to suppress showing that kinase activity is essential for suppression and suggesting that the observed suppression involves a downstream protein phosphorylation event.

Suppression of the arc35-1 mutant is probably specific to this subunit and this particular function because other mutants in Arp2/3 complex subunits, which are not defective in a cell cycle event, could not be suppressed by overexpressing CKII subunits. The same was found for overexpression of Cmd1p, which could only suppress ts growth of arc35-1 strains, but not of other mutants in the Arp2/3 complex [13]. Overexpression of CKII could not suppress the actin/endocytic defect of arc35-1, an additional criterion for specificity. These findings and previous mutant analysis suggest that various subunits of the Arp2/3 complex might fulfill both overlapping and unique functions. However, it will be interesting to see whether additional, novel arc35, arp2, and arp3 mutants will show different suppression phenotypes than the ones observed in this study.

Phenotypic similarities led us to investigate possible genetic interactions between ARC35, CKII, CMD1, and TUB4. Ts growth of the arc35-1 mutant could be suppressed by overexpression of CKII, but not its kinase-dead alleles, by Cmd1p, Cmd1-226p, and Cmd1-231p, but not by Cmd1-228p or Cmd1-239p [13], and by overexpression of Tub4p. No suppression by any of these proteins was observed when using the arp2-1 mutant. The mitotic defect of a cka2-13 strain was found to be suppressible by overexpression of Cmd1p and all cmd1 mutant alleles except Cmd1-239p, and by overexpression of Tub4p. Cmd1p or Tub4p could not suppress ts growth of the cka1-13 mutant. High levels of Tub4p suppressed the mitotic arrest of the cmd1-239 mutant. No suppression of the ts growth of other cmd1 mutants was achieved when overexpressing this series of genes. Neither was the defect of the tub4-1 mutant suppressible.

These genetic data lead to three conclusions. First, they suggests that Arc35p, CKII, Cmd1p, and Tub4p act together in a common pathway. Second, the specificity of suppression and non-suppression indicates that the pathway involves the functions defective in arc35-1, cka2-13, and cmd1-239 mutants. It is striking and revealing that all mutants suppressed undergo complete or partial metaphase arrest. Third, ts growth of the arc35-1 mutant was not suppressed by high doses of Cka1-13p and Cmd1-228p [13], which suppressed the mitotic defect of the cka2-13 strain. Besides the mitotic defect, the arc35-1 mutant is therefore defective in at least one additional essential function.

The genetic evidence cited above suggests that Arp2/3, calmodulin, CKII, and Tub4 act in a common protein kinase pathway. Our results would suggest that this protein kinase pathway is mainly localized in the cytosol. We have provided biochemical evidence for interactions between Arc35p, CKII, Cmd1p, and Tub4p in the cytosol detectable in the presence of calcium, conditions that are known to stabilize Arc35p–Cmd1p interaction [13]. These interactions are not observed in the cmd1-239 mutant, but are detectable in the cmd1-231 mutant. This confirms the specificity of the interaction and, in agreement with the genetic data, highlights the importance of the function defective in Cmd1-239p. Specificity of the observed interactions is further supported by the finding that the α-Cmd1p antibody failed to co-immunoprecipitate other proteins involved in SPB function with Cmd1p (data not shown). Based on cofractionation data of Arc35p with Arp3p, we can conclude that Arc35p associates as a subunit of the Arp2/3 complex with CKII, and most likely, Tub4p, probably through calmodulin. These interactions could be simultaneous and transient or a more stable complex could be formed. Our data cannot differentiate between these possibilities. Therefore, the Arp2/3 complex associates with Cmd1p, CKII, and Tub4p for execution of Arc35p's mitotic function.

As discussed above, the data point towards a cytosolic network of interactions involving the Arp2/3 complex, CKII, Cmd1p, and Tub4p. Genetic data indicate that a phosphorylation event by Cka2p is required for successful regulation even though the target is unknown. Cmd1p is a possible candidate.

What could be the final target of the observed interaction of the Arp2/3 complex, CKII, Cmd1p, and Tub4p? Based on phenotypic similarities, the above-mentioned proteins could regulate via Tub4p one of the mitotic functions of the spindle pole body inner plaque protein Spc110p. In support of this idea there is preliminary evidence for genetic interaction between SPC110 and ARC35/CKII/CMD1/TUB4 (our unpublished observation). It has also been demonstrated that mutations in the N-terminus of Spc110p facing the nucleoplasm lead to metaphase arrest [55]. In addition, the γ-tubulin complex is known to bind to the N-terminus of Spc110p [56–58]. Alternatively, the interaction of the Arp2/3 complex with CKII, Cmd1p, and Tub4p could be involved in functional interaction of γ-tubulin with the spindle pole body outer plaque.

Acknowledgements

We thank members of the Riezman laboratory for discussions, K. D’Hondt, F. Schaerer, and R. Lombardi for critical reading of the manuscript, A. Heese-Peck and T. Schmelzle for help with enrichment of nuclei, A. Alconada for help with the glycerol gradient, M. Hall for antibodies, D. Botstein, E. Schiebel, R. Li, and B. Winsor for strains, F. Schaerer, S. Desrivières, E. Schiebel, M. Snyder, and T. Davis for plasmids. We acknowledge the excellent technical assistance of T. Eberle, T. Aust, and J. Holenstein. This work was supported by the Canton Basel-Stadt and by a grant from the Swiss National Science Foundation to H.R.

References

[1]
Machesky
L.M.
(
1997
)
Cell motility: complex dynamics at the leading edge
.
Curr. Biol.
 
7
,
164
167
.
[2]
Machesky
L.M.
Gould
K.L.
(
1999
)
The Arp2/3 complex: a multifunctional actin organizer
.
Curr. Opin. Cell Biol.
 
11
,
117
121
.
[3]
Machesky
L.M.
et al.   (
1997
)
Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins
.
Biochem. J.
 
328
,
105
112
.
[4]
Kelleher
J.F.
Atkinson
S.J.
Pollard
T.D.
(
1995
)
Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba
.
J. Cell Biol.
 
131
,
385
397
.
[5]
Moreau
V.
Madania
A.
Martin
R.P.
Winsor
B.
(
1996
)
The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton
.
J. Cell Biol.
 
134
,
117
132
.
[6]
Welch
M.D.
DePace
A.H.
Verma
S.
Iwamatsu
A.
Mitchison
T.J.
(
1997
)
The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly
.
J. Cell Biol.
 
138
,
375
384
.
[7]
Winter
D.
Podtelejnikov
A.V.
Mann
M.
Li
R.
(
1997
)
The complex containing actin-related proteins Arp2 and Arp3 is required for the motility and integrity of yeast actin patches
.
Curr. Biol.
 
7
,
519
529
.
[8]
Moreau
V.
Galan
J.M.
Devilliers
G.
Haguenauer-Tsapis
R.
Winsor
B.
(
1997
)
The yeast actin-related protein Arp2p is required for the internalization step of endocytosis
.
Mol. Biol. Cell
 
8
,
1361
1375
.
[9]
Morrell
J.L.
Morphew
M.
Gould
K.L.
(
1999
)
A mutant of Arp2p causes partial disassembly of the Arp2/3 complex and loss of cortical actin function in fission yeast
.
Mol. Biol. Cell
 
10
,
4201
4215
.
[10]
Mullins
R.D.
Heuser
J.A.
Pollard
T.D.
(
1998
)
The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments
.
Proc. Natl. Acad. Sci. USA
 
95
,
6181
6186
.
[11]
Amann
K.J.
Pollard
T.D.
(
2001
)
The Arp2/3 complex nucleates actin filament branches from the sides of pre-existing filaments
.
Nat. Cell Biol.
 
3
,
306
310
.
[12]
Yan
C.
Leibowitz
N.
Melese
T.
(
1997
)
A role for the divergent actin gene, ACT2, in nuclear pore structure and function
.
EMBO J.
 
16
,
3572
3586
.
[13]
Schaerer-Brodbeck
C.
Riezman
H.
(
2000
)
Functional interactions between the p35 subunit of the Arp2/3 complex and calmodulin in yeast
.
Mol. Biol. Cell
 
11
,
1113
1127
.
[14]
Schaerer-Brodbeck
C.
Riezman
H.
(
2000
)
Saccharomyces cerevisiae Arc35p works through two genetically separable calmodulin functions to regulate the actin and tubulin cytoskeletons
.
J. Cell Sci.
 
113
,
521
532
.
[15]
Davis
T.N.
Urdea
M.S.
Masiarz
F.R.
Thorner
J.
(
1986
)
Isolation of the yeast calmodulin gene: calmodulin is an essential protein
.
Cell
 
47
,
423
431
.
[16]
Ohya
Y.
Ohsumi
Y.
Anraku
Y.
(
1984
)
Genetic study of the role of calcium ions in the cell division cycle of Saccharomyces cerevisiae: a calcium-dependent mutant and its trifluoperazine-dependent pseudorevertants
.
Mol. Gen. Genet.
 
193
,
389
394
.
[17]
Davis
T.N.
(
1992
)
A temperature-sensitive calmodulin mutant loses viability during mitosis
.
J. Cell Biol.
 
118
,
607
617
.
[18]
Sun
G.H.
Hirata
A.
Ohya
Y.
Anraku
Y.
(
1992
)
Mutations in yeast calmodulin cause defects in spindle pole body functions and nuclear integrity
.
J. Cell Biol.
 
119
,
1625
1639
.
[19]
Chafouleas
J.G.
Bolton
W.E.
Hidaka
H.
Boyd III
A.E.
Means
A.R.
(
1982
)
Calmodulin and the cell cycle: involvement in regulation of cell-cycle progression
.
Cell
 
28
,
41
50
.
[20]
Keith
C.H.
(
1987
)
Effect of microinjected calcium-calmodulin on mitosis in PtK2 cells
.
Cell Motil. Cytoskeleton
 
7
,
1
9
.
[21]
Rasmussen
C.D.
Means
A.R.
(
1989
)
Calmodulin is required for cell-cycle progression during G1 and mitosis
.
EMBO J.
 
8
,
73
82
.
[22]
Meggio
F.
Brunati
A.M.
Pinna
L.A.
(
1987
)
Polycation-dependent, Ca2+-antagonized phosphorylation of calmodulin by casein kinase-2 and a spleen tyrosine protein kinase
.
FEBS Lett.
 
215
,
241
246
.
[23]
Nakajo
S.
Masuda
Y.
Nakaya
K.
Nakamura
Y.
(
1988
)
Determination of the phosphorylation sites of calmodulin catalyzed by casein kinase 2
.
J. Biochem. (Tokyo)
 
104
,
946
951
.
[24]
Chen-Wu
J.L.
Padmanabha
R.
Glover
C.V.
(
1988
)
Isolation, sequencing, and disruption of the CKA1 gene encoding the alpha subunit of yeast casein kinase II
.
Mol. Cell. Biol.
 
8
,
4981
4990
.
[25]
Padmanabha
R.
Chen-Wu
J.L.
Hanna
D.E.
Glover
C.V.
(
1990
)
Isolation, sequencing, and disruption of the yeast CKA2 gene: casein kinase II is essential for viability in Saccharomyces cerevisiae
.
Mol. Cell. Biol.
 
10
,
4089
4099
.
[26]
Bidwai
A.P.
Reed
J.C.
Glover
C.V.
(
1995
)
Cloning and disruption of CKB1, the gene encoding the 38-kDa beta subunit of Saccharomyces cerevisiae casein kinase II (CKII). Deletion of CKII regulatory subunits elicits a salt-sensitive phenotype
.
J. Biol. Chem.
 
270
,
10395
10404
.
[27]
Reed
J.C.
Bidwai
A.P.
Glover
C.V.
(
1994
)
Cloning and disruption of CKB2, the gene encoding the 32-kDa regulatory beta′-subunit of Saccharomyces cerevisiae casein kinase II
.
J. Biol. Chem.
 
269
,
18192
18200
.
[28]
Sherman
S.
Fink
G.
Lawrence
C.
(
1974
)
Methods in Yeast Genetics
 .
Cold Spring Harbor Laboratory Press
,
Cold Spring Harbor, NY
.
[29]
Hicke
L.
Zanolari
B.
Pypaert
M.
Rohrer
J.
Riezman
H.
(
1997
)
Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein
.
Mol. Biol. Cell
 
8
,
13
31
.
[30]
Gietz
D.
St Jean
A.
Woods
R.A.
Schiestl
R.H.
Improved method for high efficiency transformation of intact yeast cells
.
Nucleic Acids Res.
 
20
(
1992
)
1425
.
[31]
Wach
A.
(
1996
)
PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae
.
Yeast
 
12
,
259
265
.
[32]
Wach
A.
Brachat
A.
Alberti-Segui
C.
Rebischung
C.
Philippsen
P.
(
1997
)
Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae
.
Yeast
 
13
,
1065
1075
.
[33]
Brachat
A.
Kilmartin
J.V.
Wach
A.
Philippsen
P.
(
1998
)
Saccharomyces cerevisiae cells with defective spindle pole body outer plaques accomplish nuclear migration via half-bridge-organized microtubules
.
Mol. Biol. Cell
 
9
,
977
991
.
[34]
Sambrook
J.
Fritsch
E.F.
Maniatis
T.
(
1989
)
Molecular Cloning: A Laboratory Manual
 ,
2nd
edn.
Cold Spring Harbor Laboratory Press
,
Cold Spring Harbor, NY
.
[35]
Dower
W.J.
Miller
J.F.
Ragsdale
C.W.
(
1988
)
High efficiency transformation of E. coli by high voltage electroporation
.
Nucleic Acids Res.
 
16
,
6127
6145
.
[36]
Gietz
R.D.
Sugino
A.
(
1988
)
New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites
.
Gene
 
74
,
527
534
.
[37]
Sikorski
R.S.
Hieter
P.
(
1989
)
A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae
.
Genetics
 
122
,
19
27
.
[38]
Stotz
A.
Linder
P.
(
1990
)
The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors
.
Gene
 
95
,
91
98
.
[39]
Schaerer
F.
Morgan
G.
Winey
M.
Philippsen
P.
(
2001
)
Cnm67p is a spacer protein of the Saccharomyces cerevisiae spindle pole body outer plaque
.
Mol. Biol. Cell
 
12
,
2519
2533
.
[40]
Rethinaswamy
A.
Birnbaum
M.J.
Glover
C.V.
(
1998
)
Temperature-sensitive mutations of the CKA1 gene reveal a role for casein kinase II in maintenance of cell polarity in Saccharomyces cerevisiae
.
J. Biol. Chem.
 
273
,
5869
5877
.
[41]
Hanna
D.E.
Rethinaswamy
A.
Glover
C.V.
(
1995
)
Casein kinase II is required for cell cycle progression during G1 and G2/M in Saccharomyces cerevisiae
.
J. Biol. Chem.
 
270
,
25905
25914
.
[42]
Dulic
V.
Egerton
M.
Elguindi
I.
Raths
S.
Singer
B.
Riezman
H.
(
1991
)
Yeast endocytosis assays
.
Methods Enzymol.
 
194
,
697
710
.
[43]
Geli
M.I.
Wesp
A.
Riezman
H.
(
1998
)
Distinct functions of calmodulin are required for the uptake step of receptor-mediated endocytosis in yeast: the type I myosin Myo5p is one of the calmodulin targets
.
EMBO J.
 
17
,
635
647
.
[44]
Hurt
E.C.
McDowall
A.
Schimmang
T.
(
1988
)
Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae
.
Eur. J. Cell Biol.
 
46
,
554
563
.
[45]
Munn
A.L.
Riezman
H.
(
1994
)
Endocytosis is required for the growth of vacuolar H(+)-ATPase-defective yeast: identification of six new END genes
.
J. Cell Biol.
 
127
,
373
386
.
[46]
Kilmartin
J.V.
Adams
A.E.
(
1984
)
Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces
.
J. Cell Biol.
 
98
,
922
933
.
[47]
Ohya
Y.
Botstein
D.
(
1994
)
Structure-based systematic isolation of conditional-lethal mutations in the single yeast calmodulin gene
.
Genetics
 
138
,
1041
1054
.
[48]
Spang
A.
Geissler
S.
Grein
K.
Schiebel
E.
(
1996
)
gamma-Tubulin-like Tub4p of Saccharomyces cerevisiae is associated with the spindle pole body substructures that organize microtubules and is required for mitotic spindle formation
.
J. Cell Biol.
 
134
,
429
441
.
[49]
Vogel
J.
Snyder
M.
(
2000
)
The carboxy terminus of Tub4p is required for gamma-tubulin function in budding yeast
.
J. Cell Sci.
 
113
,
3871
3882
.
[50]
Spang
A.
Grein
K.
Schiebel
E.
(
1996
)
The spacer protein Spc110p targets calmodulin to the central plaque of the yeast spindle pole body
.
J. Cell Sci.
 
109
,
2229
2237
.
[51]
Ayscough
K.R.
Stryker
J.
Pokala
N.
Sanders
M.
Crews
P.
Drubin
D.G.
(
1997
)
High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A
.
J. Cell Biol.
 
137
,
399
416
.
[52]
Rout
M.P.
Kilmartin
J.V.
(
1990
)
Components of the yeast spindle and spindle pole body
.
J. Cell Biol.
 
111
,
1913
1927
.
[53]
Evangelista
M.
et al.   (
2000
)
A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex
.
J. Cell Biol.
 
148
,
353
362
.
[54]
Geli
M.I.
Lombardi
R.
Schmelzl
B.
Riezman
H.
(
2000
)
An intact SH3 domain is required for myosin I-induced actin polymerization
.
EMBO J.
 
19
,
4281
4891
.
[55]
Sundberg
H.A.
Davis
T.N.
(
1997
)
A mutational analysis identifies three functional regions of the spindle pole component Spc110p in Saccharomyces cerevisiae
.
Mol. Biol. Cell
 
8
,
2575
2590
.
[56]
Knop
M.
Schiebel
E.
(
1997
)
Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p
.
EMBO J.
 
16
,
6985
6995
.
[57]
Nguyen
T.
Vinh
D.B.
Crawford
D.K.
Davis
T.N.
(
1998
)
A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and Spc98p, components of the yeast gamma-tubulin complex
.
Mol. Biol. Cell
 
9
,
2201
2216
.
[58]
Elliott
S.
Knop
M.
Schlenstedt
G.
Schiebel
E.
(
1999
)
Spc29p is a component of the Spc110p subcomplex and is essential for spindle pole body duplication
.
Proc. Natl. Acad. Sci. USA
 
96
,
6205
6210
.

Author notes

1
ESBATech AG, 8952 Zurich-Schlieren, Switzerland.