KEY RESOURCES TABLE.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Rat monoclonal anti-HA (clone 3F10) | Roche | Cat#11867423001 (Lot#42155800) |
| Mouse monoclonal anti-FLAG M2 | Sigma | Cat#F1804 (Lot#SLBJ4607V) |
| Rabbit polyclonal anti-HA | AbClonal | Cat#AE036 (Lot#356155811) |
| Rabbit polyclonal anti-FLAG | Sigma | Cat#F7425 |
| Mouse anti-HA | Santa Cruz | Cat#SC-7392 |
| Mouse anti-FLAG (clone M2) | Sigma | Cat#F-1804 |
| Mouse monoclonal anti-α-tubulin (B512) | Sigma | Cat#T6074 (Lot#037M4804V) |
| Goat anti-rat IgG HRP | EMD Millipore | Cat#AP136 (Lot#3270737) |
| Goat anti-mouse IgG HRP | EMD Millipore | Cat#12-349 (Lot#2985412) |
| Alexa Fluor 488 Goat anti-rat IgG (H+L) | Invitrogen | Cat#A11006 (Lot#2299157) |
| Alexa Fluor 594 Goat anti-mouse IgG (H+L) | Invitrogen | Cat#A11005 (Lot#1830459) |
| Alexa Fluor 488 Donkey Anti-Rat | Invitrogen | Cat#A48269 |
| Alexa Fluor 594 Donkey anti-mouse | Invitrogen | Cat#A32744 |
| Mouse monoclonal anti-GST | Santa Cruz | Cat#SC-138 |
| Rabbit polyclonal anti-MAR1 (Custom-generated against MAR1 peptide: TQPPRPPWPPRPPPAPPPS, residues 164–182) | YenZym™ | Rabbit#YZ5485-6 (Lot#50317) |
| Bacterial and virus strains | ||
| B21 (DE3) cells | New England Biolabs | Cat#C2527I |
| Chemicals, peptides, and recombinant proteins | ||
| Protease Inhibitor cocktail for Plant cell and tissue extracts | Roche | Cat#11836153001 (Lot#25765800) |
| Poly-l-lysine | Sigma | Cat#P8920 |
| Fluoromount-G | Southern Biotech | Cat#0100-01 (Lot#D1810-Q490) |
| Prolong Gold antifade reagent | Invitrogen | Cat#P36934 (Lot#2322656) |
| SuperSignal West Femto Max Sensitivity substrate | ThermoFisher Scientific | Cat#34095 (Lot#WE325003) |
| Protein A Sepharose 4B Fast Flow from S. aureus | Sigma | Cat#P9424 (Lot#MKBZ075IV) |
| lysozyme | ThermoFisher Scientific | Cat#89833 |
| IPTG | ThermoFisher Scientific | Cat#34060 |
| HRP-conjugated streptavidin | Jackson ImmunoResearch | Cat#016030084 |
| Paraformaldehyde | Aldrich | Cat#441244 (Lot#MKCB6246) |
| Phire Hot Start II DNA Polymerase | ThermoFisher Scientific | Cat#F-122 (Lot#00760841) |
| Chelex 100 Resin | BioRad | Cat#142-1253 (Lot #: 64404479) |
| Agar, microbiology tested suitable for plant cell culture | Sigma | Cat#A1296 (Lot#BCCD6437) |
| 30% Acrylamide/Bis Solution 37.5:1 | BioRad | Cat#1610158 (Lot#64316706) |
| Ammonium persulfate | Sigma | Cat#A3678 (Lot#MKBW1233V) |
| N,N,N’,N’- Tetramethyl ethylenediamine (TEMED) | Sigma | Cat#T9281 (Lot#BCCF2825) |
| TCEP-HCl | GoldBio | Cat#TCEP10 (Lot#9915.052616A) |
| DTT | Sigma | Cat#D0632 (Lot#SLBW1508) |
| HgCl2 | ThermoFisher Scientific | Cat#M155I |
| OsO4 | Electron Microscopy Sciences | Cat#19140 |
| Glutaraldehyde solution, grade II, 25% in H2O | Sigma | Cat#G-6257 (Lot#55H0619) |
| Dibutyryl-cAMP | Sigma | Cat#D0627 |
| EZ-Link™ Sulfo-NHS-LC-Biotin | ThermoFisher Scientific | Cat#21335 (Lot#QJ222159) |
| Pronase | Roche | Cat#10165921001 (Lot#70506922) |
| Trypsin from bovine pancreas | Sigma | Cat#T8003 (Lot#SLBM232IV) |
| Trypsin inhibitor from chicken egg white | Sigma | Cat#T2011 (Lot#SLBP7284V) |
| Methanol | Pharmco | Cat#339000000 (Lot#C19C12006) |
| Sodium orthovanadate (Na3O4V) | Aldrich | Cat#450243 (Lot#MKBZ693OV) |
| NaF | Sigma | Cat#201154 |
| Phenylmethylsulfonylfluoride (PMSF) | Roche | Cat#15460420 (Lot#10837091001) |
| Gelatin from cold water fish skin | Sigma | Cat#G7765 (Lot#SLBS7921) |
| Goat Serum | Sigma | Cat#G9023 (Lot#SLCB4567) |
| Donkey Serum | Sigma | Cat#D9663 (Lot#SLCD2393) |
| Sodium deoxycholate | Sigma | Cat#D6750 (Lot#126K0061) |
| Triton X-100 | Sigma | Cat#X-100 (Lot#SLBM7930V) |
| Transcriptor First Strand cDNA Synthesis kit | Roche | Cat#04379012001 (Lot#10769023) |
| DNase I | Ambion | Cat#AM2222 |
| RNase A | Thermo Scientific | Cat#EN0531 |
| Glutathione beads | Sigma | Cat#G4510 |
| Glutathione, reduced | Sigma | Cat#G6013 |
| Infusion Dry-down PCR Cloning Kit | Takara (Clontech) | Cat#639602 |
| HA peptide | Roche | Cat#11666975001 |
| FLAG peptide | Sigma | Cat#F4799 |
| Experimental models: Organisms/strains | ||
| C. reinhardtii: LMJ.RY0402.185187(−) or mar1(−): Cre03.g176961::pCIB(−) | CLiP Library (Li et al., 2019) https://www.chlamylibrary.org/index | LMJ.RY0402.185187 |
| C. reinhardtii: progeny 106: mar1(−) | This paper | N/A |
| C. reinhardtii: progeny 133: mar1(−) | This paper | N/A |
| C. reinhardtii: progeny 144: mar1(−) | This paper | N/A |
| C. reinhardtii: WT(−): cw15(−) | Chlamydomonas Resource Center https://www.chlamycollection.org/ | CC-5325 |
| C. reinhardtii: CC-5313: Cre12g541400::pSL72(+) | Chlamydomonas Resource Center | CC-5313 |
| C. reinhardtii: progeny 3d-1: mar1;MAR1-FLAG(+) | This paper | N/A |
| C. reinhardtii: MF-c7: mar1::MAR1-FLAG(−) | This paper | N/A |
| C. reinhardtii: progeny 111: mar1;MAR1-FLAG(−) | This paper | N/A |
| C. reinhardtii: progeny 115: mar1;MAR1-FLAG(−) | This paper | N/A |
| C. reinhardtii: progeny 147: mar1;MAR1-FLAG(−) | This paper | N/A |
| C. reinhardtii: progeny 73: mar1;hap2(−) | This paper | N/A |
| C. reinhardtii: progeny 98: mar1;hap2(−) | This paper | N/A |
| C. reinhardtii: progeny 148: mar1;hap2(+) | This paper | N/A |
| C. reinhardtii: progeny 156: mar1;hap2(+) | This paper | N/A |
| C. reinhardtii: FUS1-HA(+): fus1::FUS1-HA(+) | (Liu et al., 2010) | N/A |
| C. reinhardtii: fus1(+): imp1-15 (fus1) | Chlamydomonas Resource Center (Ferris et al., 1996) | CC-1158 |
| C. reinhardtii: HAP2-FLAG(−): hap2[40d4]::HAP2-FLAG-ble(−) | Chlamydomonas Resource Center (Liu et al., 2010) | CC-5309 |
| C. reinhardtii: HAP2-HA(−) or WT(−): hap2[40d4]::HAP2-HA(−) | Chlamydomonas Resource Center (Feng et al., 2018; Liu et al., 2015) | CC-5295 |
| C. reinhardtii: hap2(−): [40d4] B215.Cre16.g674852::pMN56(−) | Chlamydomonas Resource Center (Liu et al., 2015) | CC-5281 |
| C. reinhardtii: WT(+): 21gr | Chlamydomonas Resource Center (Proschold et al., 2005; Sager, 1955) | CC-1690 |
| C. reinhardtii: IFT172-FLAG: fla11-2; FLAG-IFT172 (1G3) | Chlamydomonas Resource Center (Lin et al., 2018) | CC-5466 |
| C. reinhardtii: IMP3-HA: imp3::IMP3-HA | Chlamydomonas Resource Center (Lin et al., 2013) | CC-5149 |
| C. reinhardtii: progeny 78: mar1;hap2;MAR1-FLAG(−) | This paper | N/A |
| C. reinhardtii: progeny 146: mar1;hap2; MAR1-FLAG(−) | This paper | N/A |
| C. reinhardtii: progeny 122: mar1;HAP2-HA (−) | This paper | N/A |
| C. reinhardtii: progeny 132: mar1;hap2;HAP2-HA(−) | This paper | N/A |
| C. reinhardtii: HF-6: mar1::HAP2-FLAG(−) | This paper | N/A |
| C. reinhardtii: progeny HF-10: mar1::HAP2-FLAG(−) | This paper | N/A |
| C. reinhardtii: hap2::HAP2-HA;MAR1-FLAG(−) | Chlamydomonas Resource Center | CC-5284 |
| C. reinhardtii: progeny 102: mar1;MAR1-FLAG;hap2;HAP2-HA(−) | This paper | N/A |
| C. reinhardtii: progeny 114: mar1;MAR1-FLAG;hap2;HAP2-HA(−) | This paper | N/A |
| C. reinhardtii: progeny 131: mar1;MAR1-FLAG;hap2;HAP2-HA(−) | This paper | N/A |
| Oligonucleotides | ||
| HSV-TEV-FLAG: CAGCCAGAACTCGCCCCGGAAGACCCCGAGGATGATCGA TCCGGACCGGAGAACCTGTATTTCCAGGGAGCCATCCCC ACGACGGAGAACCTGTACTTCCAGGTGGACGCCAACTGC CGGCCCGGCTCCTCCATGGACTACAAGGACCACGATGGC GATTACAAGGATCACGACATCGACTACAAGGACGACGAC GACAAG |
This paper | N/A |
| p1 Mar1_185187_F2 genotyping: CACCGCCTGGAACTACTCCTC |
This paper | N/A |
| p2 Mar1_185187_R2 genotyping: TTGGGTCTGGTTTAGATTTGGGCTGG |
This paper | N/A |
| RB1 CC-5313 genotyping: ATGGGGCGGTATCGGAGGAAAAG |
This paper | N/A |
| C12g541400.R2 CC-5313 genotyping: TGTAGCAAGGCTCCCGCTTCTAGTTG |
This paper | N/A |
| Zeo_F1 zeocin genotyping: GCTGCATGTGCACAGTCACGCTGTCTC |
This paper | N/A |
| Zeo_R1 zeocin genotyping: GGATCCCACACACCTGCCCGTCT |
This paper | N/A |
| Fus1_P3_Fw plus gamete genotyping: GACCATCGTAGAGCGCTCTCACCAATTG |
This paper | N/A |
| Fus1_P3_Rev plus gamete genotyping: CCGCTACGCTTCTGCGTCTTGATAGTC |
This paper | N/A |
| Nit_p1_F hap2-2 genotyping: GGTCGAGGTGCCGTAAAGC |
This paper | N/A |
| Nit_p2_R hap2-2 genotyping: CCTGATGCCCCTTCAACCG |
This paper | N/A |
| L8 MAR1 gene amplification: CGGCGGCTTTGTTCCGTTTGTTTG |
This paper | N/A |
| R5B MAR1 gene amplification: CAGTCTGCGTTTCCCTTCTCGTGG |
This paper | N/A |
| G176961.R20 ble insertion into MAR1-FLAG: CCGCGAATTCactagtGCTGCATGTGCACAGTCACGC |
This paper | N/A |
| L20 ble insertion into MAR1-FLAG: GCCCCGTGCTcctaggGGATCCCACACACCTGCCC |
This paper | N/A |
| PGenD.s ble insertion into HAP2-FLAG: actagtGGATCCCACACACCTGCCCGTCTG |
This paper | N/A |
| PGenD.as ble insertion into HAP2-FLAG: tctagaGCTGCATGTGCACAGTCACGCTGTCT |
This paper | N/A |
| g176961-L3 HIS-MAR1: TCCGGACTTGCCTTTTTAGC |
This paper | N/A |
| g176961-R10 HIS-MAR1: GGCTTCCTACAGGCCCACCA |
This paper | N/A |
| Recombinant DNA | ||
| Plasmid. pGenD-Ble | Chlamydomonas Resource Center (Fischer and Rochaix, 2001) | https://www.chlamycollection.org/ |
| pGEM-T-easy vector, MAR1 | Promega | Cat#A1360 (Lot#219942) |
| PET28a(+) vector, HIS-MAR1 | GenScript assisted in cloning, see Supplemental Data S3 | N/A |
| Plasmid. HAP2-HA: pYJ36 | (Liu et al., 2008) | N/A |
| Plasmid. HAP2- FLAG: C2925/pYJHSVTEVFL-zeo | This paper | N/A |
| Plasmid. pGEX-2T empty vector | Sigma | Cat#GE28-9546-53 |
| Plasmid. GST-rFUS1: pGEX-2T FUS1 | (Misamore et al., 2003) | N/A |
| Plasmid. MAR1-FLAG: DH5 α/pYJFUS1BHSVFL+Z | This paper | N/A |
| Software and algorithms | ||
| Leica LAS X core module | Leica Microsystem Inc. | https://www.leica-microsystems.com |
| StreamPix 5.8.1.0 | NorPix | https://www.norpix.com/products/streampix/streampix.php |
| GraphPad Prism Version 9.0.0 | GraphPad | https://www.graphpad.com/ |
| Image Studio™ | LI-COR | https://www.licor.com/bio/image-studio/ |
| PyMol Software | Schrodinger, LLC | https://www.schrodinger.com/products/pymol |
| Adobe Illustrator and Photoshop | Adobe | https://www.adobe.com/products/catalog.html |
| RaptorX | (Källberg et al., 2012; Xu, 2019; Xu et al., 2021) | http://raptorx.uchicago.edu/ |
| PROMALS3D | (Pei and Grishin, 2007) | http://prodata.swmed.edu/promals3d/promals3d.php |
| DELTA-BLAST | (Boratyn et al., 2012) | https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROGRAM=blastp&BLAST_PROGRAMS=deltaBlast |
| PSI-BLAST | (Altschul et al., 1997; Aravind and Koonin, 1999) | https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROGRAM=blastp&BLAST_PROGRAMS=psiBlast |
| PHYRE2 | (Kelley et al., 2015) | http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index |
| SWISS-MODEL | (Waterhouse et al., 2018) | https://swissmodel.expasy.org/ |
| HHPRED | (Zimmermann et al., 2018) | https://toolkit.tuebingen.mpg.de/tools/hhpred |