A Humanized Yeast Model for Studying TRAPP Complex Mutations; Proof-of-Concept Using Variants from an Individual with a TRAPPC1-Associated Neurodevelopmental Syndrome
Abstract
:1. Introduction
2. Materials and Methods
2.1. Preparation of sgRNAs That Target the Yeast TRAPP Genes
2.2. Preparation of Repair Template DNA
2.3. Yeast Transformations
2.4. Primer Design for Confirmation via Colony PCR
2.5. Colony Screening via PCR
2.6. Yeast Liquid Growth Assays
2.7. Autophagy Assays in Yeast
2.8. Preparation of Yeast Cell Lysates
2.9. Invertase Secretion Assay
2.10. Western Blot Analysis
2.11. Genetic Studies
2.12. Cell Culture
2.13. Autophagy Assays in Mammalian Cells
2.14. Antibodies and Dyes
2.15. Immunofluorescence Microscopy
2.16. Retention Using Selective Hooks (RUSH) Assay
3. Results
3.1. TRAPPC1, TRAPPC2, TRAPPC2L, TRAPPC6A, and TRAPPC6B Successfully Replace Their Yeast Orthologs BET5, TRS20, TCA17, and TRS33, Respectively
3.2. TRAPPC3, TRAPPC4, and TRAPPC5 Cannot Complement the Loss of BET3, TRS23, and TRS31, Respectively, in Yeast
3.3. Humanized Yeast Strains Show a Slower Growth Phenotype Compared to the Parental Yeast Strains
3.4. A Novel Disorder Associated with Biallelic TRAPPC1 Variants
3.5. Humanized Yeast as a Platform for Characterizing the First Reported Mutation in TRAPPC1
3.6. Humanized Yeast with C1 (c.64_72del) Is Conditional-Lethal
3.7. Non-Selective Autophagy Is Defective in the Humanized C1 (c.64_72del) Yeast Strain
3.8. Secretion Is Defective in the Humanized C1 (c.64_72del) Yeast Strain
3.9. Fibroblasts from the Affected Individual with TRAPPC1 Mutations Do Not Show an Accumulation of LC3-II during Starvation
3.10. Wild-Type TRAPPC1 Rescues a Membrane Trafficking Defect and Golgi Fragmentation in Patient Fibroblasts
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Mammalian Subunits | Yeast Subunits | TRAPP Complex | Essential in Yeast? | %Identity/Similarity of Core Subunits |
---|---|---|---|---|
TRAPPC1 | Bet5p | II/III | yes | 33/54 |
TRAPPC2 | Trs20p | II/III | yes | 34/52 |
TRAPPC2L | Tca17p | II | no | 24/43 |
TRAPPC3 | Bet3p | II/III | yes | 56/76 |
TRAPPC4 | Trs23p | II/III | yes | 29/44 |
TRAPPC5 | Trs31p | II/III | yes | 31/52 |
TRAPPC6A/C6B | Trs33p | II/III | no | 35/51 and 33/47 |
TRAPPC8 | Trs85p | III | no | - |
TRAPPC9 | Trs120p | II | yes | - |
TRAPPC10 | Trs130p | II | yes | - |
TRAPPC11 | - | III | - | - |
TRAPPC12 | - | III | - | - |
TRAPPC13 | Trs65 | III/II * | no | - |
Plasmid | Construct or Backbone |
---|---|
MSB2 | pRS315 |
MSB206 | pRS313 |
MSB230 | pRS313-TRS20 |
MSB1253 | pRS416-GFP-ATG8 |
MSB1790 | pmRFP-N1-TRAPPC1 |
MSB1791 | pmRFP-C1-TRAPPC1 |
MSB1797 | pRS413-ADH1-TRAPPC1 |
MSB1800 | pRS413-ADH1-TRAPPC1 (c.362_363del) |
MSB1801 | pRS413-ADH1-TRAPPC1 (c.64_72del) |
MSB1805 | pCEN6-Cas9-GFP-KanMX |
MSB1806 | pCEN6-Cas9-GFP-KanMX BET5 sgRNA1 |
MSB1820 | pCEN6-Cas9-GFP-KanMX TRS20 sgRNA1 |
MSB1821 | pCEN6-Cas9-GFP-KanMX TRS23 sgRNA1 |
MSB1823 | pCEN6-Cas9-GFP-KanMX BET3 sgRNA1 |
MSB1824 | pCEN6-Cas9-GFP-KanMX TRS31 sgRNA1 |
MSB1826 | pCEN6-Cas9-GFP-KanMX TCA17 sgRNA1 |
MSB1829 | pCEN6-Cas9-GFP-KanMX TRS33 sgRNA1 |
MSB1839 | pRS413-ADH1-TRAPPC5 |
MSB1840 | pRS413-GPD-TRAPPC5 |
MSB1841 | pRS423-GPD-TRAPPC5 |
MSB1855 | pRS413-ADH1-TRAPPC3 |
MSB1856 | pRS413-GPD-TRAPPC3 |
MSB1857 | pRS423-GPD-TRAPPC3 |
MSB1858 | pRS413-ADH1-TRAPPC4 |
MSB1859 | pRS413-GPD-TRAPPC4 |
MSB1860 | pRS423-GPD-TRAPPC4 |
Yeast Strain | Genotype | Source |
---|---|---|
MSY61 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 bet3∆::KanMX pRS315-BET3 | Sacher Lab |
MSY62 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs23∆::KanMX pRS316-TRS23 | Sacher Lab |
MSY64 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 bet5∆::KanMX pRS316-BET5 | Sacher Lab |
MSY65 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs31∆::KanMX pRS316-TRS31 | Sacher Lab |
MSY135 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 | Sacher Lab |
MSY362 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 trs85∆::KanMX | Titorenko Lab |
MSY563 | MATα his3Δ1 leu2Δ0 ura3ΔMET15 trs23Δ::KanMX pRS315-TRS23 TRS85–3xHA::HIS3 | Sacher Lab |
MSY706 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 atg1∆::KanMX | Brett Lab |
MSY710 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 atg1∆::KanMX pRS416-GFP-ATG8 | Sacher Lab |
MSY740 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 trs85∆::KanMX pRS416-GFP-ATG8 | Sacher Lab |
MSY766 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 pRS416-GFP-ATG8 | Sacher Lab |
MSY901 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 TRS130-3xHA::HIS3 | Sacher Lab |
MSY950 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 bet5∆::KanMX pRS316-BET5 pRS413-ADH1 | Zykaj E. |
MSY951 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 bet5∆::KanMX pRS316-BET5 pRS413-ADH1-TRAPPC1 | Zykaj E. |
MSY952 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 bet5∆::KanMX pRS316-BET5 pRS313-BET5 | Zykaj E. |
MSY955 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 bet5∆ with TRAPPC1 | Zykaj E. |
MSY956 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 bet5∆ with TRAPPC1 ∆HRK (c.64_72del) | Zykaj E. |
MSY957 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 trs20∆ with TRAPPC2 | Zykaj E. |
MSY958 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 TRS130-3xHA::HIS3 tca17∆ with TRAPPC2L | Zykaj E. |
MSY959 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 TRS130-3XHA::HIS3 trs33∆ with TRAPPC6A | Zykaj E. |
MSY960 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 TRS130-3XHA::HIS3 trs33∆ with TRAPPC6B | Zykaj E. |
MSY963 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs31∆::KanMX pRS316-TRS31 pRS413-ADH1-TRAPPC5 | Zykaj E. |
MSY964 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs31∆::KanMX pRS316-TRS31 pRS413-GPD-TRAPPC5 | Zykaj E. |
MSY965 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs31∆::KanMX pRS316-TRS31 pRS423-GPD-TRAPPC5 | Zykaj E. |
MSY967 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs31∆::KanMX pRS316-TRS31 pRS315 | Zykaj E. |
MSY968 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs31∆::KanMX pRS316-TRS31 pRS315-TRS31 | Zykaj E. |
MSY971 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs23∆::KanMX pRS316-TRS23 pRS313 | Zykaj E. |
MSY972 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs23∆::KanMX pRS316-TRS23 pRS313-TRS23 | Zykaj E. |
MSY973 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs23∆::KanMX pRS316-TRS23 pRS413-ADH1-TRAPPC4 | Zykaj E. |
MSY974 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs23∆::KanMX pRS316-TRS23 pRS413-GPD-TRAPPC4 | Zykaj E. |
MSY975 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 trs23∆::KanMX pRS316-TRS23 pRS423-GPD-TRAPPC4 | Zykaj E. |
MSY976 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 bet3∆::KanMX pRS315-BET3 pRS313 | Zykaj E. |
MSY977 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 bet3∆::KanMX pRS315-BET3 pRS313–BET3 | Zykaj E. |
MSY978 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 bet3∆::KanMX pRS315-BET3 pRS413-ADH1-TRAPPC3 | Zykaj E. |
MSY979 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 bet3∆::KanMX pRS315-BET3 pRS413-GPD-TRAPPC3 | Zykaj E. |
MSY980 | MATα his3Δ1 leu2Δ0 ura3Δ0 MET15 bet3∆::KanMX pRS315-BET3 pRS423-GPD-TRAPPC3 | Zykaj E. |
MSY981 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 bet5∆ with TRAPPC1 pRS416-GFP-ATG8 | Zykaj E. |
MSY982 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 bet5∆ with TRAPPC1 ∆HRK (c.64_72del) pRS416-GFP-ATG8 | Zykaj E. |
MSY983 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 TRS130-3xHA::HIS3 bet5∆ with TRAPPC1 | Zykaj E. |
MSY984 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 TRS130-3xHA::HIS3 bet5∆ with TRAPPC1 ∆HRK (c.64_72del) | Zykaj E. |
MSY987 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 tca17∆ with TRAPPC2L | Zykaj E. |
MSY988 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 trs33∆ with TRAPPC6B | Zykaj E. |
MSY993 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 trs33∆ with TRAPPC6A | Zykaj E. |
Primer Name | Sequence |
---|---|
BET5 sgRNA1-Fp | GACTTTTTACACAAAAGCTCTCCAAA |
BET5 sgRNA1-Rp | AAACTTTGGAGAGCTTTTGTGTAAAA |
TRS20 sgRNA1-Fp | GACTTTACCAATGCAGAAAATCCACA |
TRS20 sgRNA1-Rp | AAACTGTGGATTTTCTGCATTGGTAA |
TCA17 sgRNA1-Fp | GACTTTGATTTACGTCCCTAATGAGG |
TCA17 sgRNA1-Rp | AAACCCTCATTAGGGACGTAAATCAA |
BET3 sgRNA1-Fp | GACTTTCTTCAAAGACCTCGATTGCG |
BET3 sgRNA1-Rp | AAACCGCAATCGAGGTCTTTGAAGAA |
TRS23 sgRNA1-Fp | GACTTTCTTGTAATAAACAAATCAGG |
TRS23 sgRNA1-Rp | AAACCCTGATTTGTTTATTACAAGAA |
TRS31 sgRNA1-Fp | GACTTTGCATCTGACCAACAATTCCC |
TRS31 sgRNA1-Rp | AAACGGGAATTGTTGGTCAGATGCAA |
TRS33 sgRNA1-Fp | GACTTTGGACCTTGTGGAGAAGATTG |
TRS33 sgRNA1-Rp | AAACCAATCTTCTCCACAAGGTCCAA |
Primer Name | Sequence |
---|---|
TRAPPC1-Fp | AATACAAAAAAAAAATACAGAACTATCGTAAGATAATCTGCTTTTGATATAAATCATAAATCAGTGGAGCATGACTGTCCACAACCTGTAC |
TRAPPC1-Rp | TATACGATGAATGCAATTCAATTCATCGCTTCTATTTATTTTAGGTTTTTCTTCTGCTCATGGTTCGTGCTCAGCCAGCCCGGGCGGAGAAG |
TRAPPC2-Fp | AAGAAAAAAAAGAGAAGGTAAACACTAATACGAATAGAGAATACAGAAAAAACATACAAAGCACATTGAGATGTCTGGGAGCTTCTACTTTG |
TRAPPC2-Rp | AAAAAAAAAAAAATACATACTACATATACATATACGCCATAAAAAATCTCTGCATCTATCTTATTTCCCATCAGCTTAAAAGGTGTTTCTTC |
TRAPPC2L-Fp | AAGTTATTGAGTTGAAAATTGAGTAAATATTCTCTCCAAAAATCAAATTGTACGTTTATATCACCAAACTATGGCGGTGTGCATCGCGGTG |
TRAPPC2L-Rp | GCTCTCACTCCAAAGCAGTGACATTAAATTTTGCTTTCATTCGTGAAAGAATCGACTAATAAATTATCATTCAGCACACCTGTATCATCATC |
TRAPPC3-Fp | CCATAGCTATAGAGATGGGAATGCAGAGTAAAGCTTCAAGTGTTCATTGATAACCAAAACTGGGTCAAAAATGTCGAGGCAGGCGAACCGTG |
TRAPPC3-Rp | AAATAATGTATACCTAAATAACGACACCAATAATAACAAAAATCACGAAAAAAAAAAGCTTACATGTCCATTATTCCTCTCCAGCTGGAAG |
TRAPPC4-Fp | AAAAGGAATCTGCCTTTGCATAAGTTCAAAAGTGCAATTTTAGTGTTGGATTTAAACGGGAAAAATTGAAATGGCGATTTTTAGTGTGTATG |
TRAPPC4-Rp | CTTAGTTCTAGAAGTACGTAGTATTTATTTTCTTGGTGTGAATGCGTTTATCTTCCGATGGCGCGTGCGTCTATGACCCAGGTCCAAAAG |
TRAPPC5-Fp | AGGACAAGAGACACGGAAGCGACAGAAAGACAGGGAGTACTATCACCATCCATAATACTGGAGTAACATTATGGAGGCGCGCTTCACGCGC |
TRAPPC5-Rp | TCAACAGTAAATATACAAACTCTTTATCCTTTTCTTGGTTTTAGGTATTTACCGATGTTTTGAGATGATATCAGCGGCCCTCCAGGGCCCG |
TRAPPC6A-Fp | AGCTGAGAGTGAAGACCTCATGCCATAAAAGAAAACATACACCGGTTATAAAGTTGGAAGATAAACAATTATGGCGGATACTGTGTTGTTTG |
TRAPPC6A-Rp | TCTTTTATATACACACTTATATCTATCTATATGTCGATGTACATTCTTAGAACAAAAATCTGTCGGACCTTTAGGATTTCGGAATCACCACC |
TRAPPC6B-Fp | AGCTGAGAGTGAAGACCTCATGCCATAAAAGAAAACATACACCGGTTATAAAGTTGGAAGATAAACAATTATGGCGGATGAGGCGTTGTTTTTG |
TRAPPC6B-Rp | TCTTTTATATACACACTTATATCTATCTATATGTCGATGTACATTCTTAGAACAAAAATCTGTCGGACCTCTACAGCTTCTGTATCATCAC |
Primer Name | Sequence |
---|---|
TRAPPC1-Fp | GTCCTTTACTATTGACTAGTG |
TRAPPC1-Rp | CATCCCGTACATCAGCTTATAC |
TRAPPC2-Fp | CCCTTTACCTCTTCAAAGCCT |
TRAPPC2-Rp | AAGTACATGTTGTTCGATAGC |
TRAPPC2L-Fp | AAGTTCTGCCGGAAGAGCTCATC |
TRAPPC2L-Rp | ATTGCGGAGATCTTCTCATCC |
TRAPPC6A-Fp | GGAATCTGGTATGCACTCAC |
TRAPPC6A-Rp | TGTTGTCTTGCAGGACGTAG |
TRAPPC6B-Fp | GGAATCTGGTATGCACTCAC |
TRAPPC6B-Rp | AACTCATCCTTGAACCTTGCAG |
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Zykaj, E.; Abboud, C.; Asadi, P.; Warsame, S.; Almousa, H.; Milev, M.P.; Greco, B.M.; López-Sánchez, M.; Bratkovic, D.; Kachroo, A.H.; et al. A Humanized Yeast Model for Studying TRAPP Complex Mutations; Proof-of-Concept Using Variants from an Individual with a TRAPPC1-Associated Neurodevelopmental Syndrome. Cells 2024, 13, 1457. https://doi.org/10.3390/cells13171457
Zykaj E, Abboud C, Asadi P, Warsame S, Almousa H, Milev MP, Greco BM, López-Sánchez M, Bratkovic D, Kachroo AH, et al. A Humanized Yeast Model for Studying TRAPP Complex Mutations; Proof-of-Concept Using Variants from an Individual with a TRAPPC1-Associated Neurodevelopmental Syndrome. Cells. 2024; 13(17):1457. https://doi.org/10.3390/cells13171457
Chicago/Turabian StyleZykaj, Erta, Chelsea Abboud, Paria Asadi, Simane Warsame, Hashem Almousa, Miroslav P. Milev, Brittany M. Greco, Marcos López-Sánchez, Drago Bratkovic, Aashiq H. Kachroo, and et al. 2024. "A Humanized Yeast Model for Studying TRAPP Complex Mutations; Proof-of-Concept Using Variants from an Individual with a TRAPPC1-Associated Neurodevelopmental Syndrome" Cells 13, no. 17: 1457. https://doi.org/10.3390/cells13171457