REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Sheep anti-Mzt1 (serum)	This paper	N/A
Sheep anti-Alp6 (serum)	Homemade lab stock	N/A
Sheep anti-Alp4 (serum)	Homemade lab stock	N/A
Sheep anti-Alp6, affinity-purified	Homemade lab stock	N/A
Sheep anti-Alp4, affinity-purified	Homemade lab stock	N/A
Mouse monoclonal anti-γ-tubulin antibody, clone GTU-88	Sigma-Aldrich	Cat# T6557; RRID:AB_477584
Sheep anti-Mto1 (serum)	Homemade lab stock	N/A
Sheep anti-Mto2 (serum)	Homemade lab stock	N/A
Sheep anti-GFP, affinity-purified	Homemade lab stock	N/A
Mouse monoclonal Anti-FLAG® M2 antibody	Sigma-Aldrich	Cat# F1804; RRID:AB_262044
Mouse monoclonal anti-Goat/Sheep IgG antibody, clone GT-34	Sigma-Aldrich	Cat# G2904; RRID:AB_259856
IRDye 800CW Donkey anti-Mouse IgG (H+L)	LI-COR	Cat# 926-32212; RRID:AB_621847
Bacterial and Virus Strains
Escherichia coli DH10 EMBacY	Imre Berger Lab [38];	N/A
Escherichia coli BL21-CodonPlus (DE3)-RIL	Agilent	Cat# 230245
Chemicals, Peptides, and Recombinant Proteins
4,6-diamidino-2-phenylindole, dihydrochloride (DAPI)	Thermo Fisher Scientific	Cat# D1306
Tubulin (porcine) > 99%	Cytoskeleton, Inc.	Cat# T240
Rhodamine-labeled porcine tubulin	Cytoskeleton, Inc.	Cat# TL590M
Chymostatin	Melford	Cat# C1104
Leupeptin hydrochloride > 90%	Apollo Scientific	Cat# BIMI2442
Aprotinin	Apollo Scientific	Cat# BIMI2132
Antipain dihydrochloride	Apollo Scientific	Cat# BIA0201
Pepstatin A	Apollo Scientific	Cat# BIMI2205
E-64	Apollo Scientific	Cat# BIMI2157
Amylose resin	New England Biolabs	Cat# E8021L
Glutathione-agarose resin	Sigma-Aldrich	Cat# G4510
Strep-Tactin Sepharose resin	IBA GmBH	Cat# 2-1201-010
Fractogel EMD Chelate	Merck	Cat# 1.10338.0010
X-tremeGENE HP DNA Transfection Reagent	Roche	Cat# 6366244001
GIBCO Express Five SFM media	Thermo Fisher Scientific	Cat# 10486-025
Sf-900 II SFM	Thermo Fisher Scientific	Cat# 10902-096
L-Glutamine 200mM	Thermo Fisher Scientific	Cat# 25030024
Cre recombinase	New England Biolabs	Cat# M0298
Dextran sulfate sodium salt	Sigma-Aldrich	Cat# 31404
SYPRO Ruby gel stain	Lonza	Cat# LZ50562
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)	Thermo Fisher Scientific	Cat# 22980
sulfosuccinimidyl 4,4’-azipentanoate (Sulfo-NHS-Diazirine) (Sulfo-SDA)	Thermo Fisher Scientific	Cat# 26173
N-hydroxysulfosuccinimide (Sulfo-NHS)	Thermo Fisher Scientific	Cat# 24525
Ammonium bicarbonate	Sigma-Aldrich	Cat# 09830
1,4-Dithiothreitol (DTT)	Sigma-Aldrich	Cat# 10197777001
Iodoacetamide	Sigma-Aldrich	Cat# I1149
Pierce™ Trypsin protease	Thermo Fisher Scientific	Cat# 90057
RunBlue™ SDS Running Buffer	Expedeon	Cat# NXB50500
Protein G Dynabeads	Thermo Fisher Scientific	Cat# 10003D
Bacto Yeast Extract	BD Biosciences	Cat# 212750
Bacto Agar	BD Biosciences	Cat# 214030
Critical Commercial Assays
Gateway® LR Clonase® II enzyme mix	Thermo Fisher Scientific	Cat# 11791-020
Gateway® BP Clonase® II enzyme mix	Thermo Fisher Scientific	Cat# 11789-020
Deposited Data
MS proteomics data deposited to ProteomeXchange Consortium via PRIDE	This paper	ProteomeXchange: PXD012624
Experimental Models: Cell Lines
Sf9 cells	Thermo Fisher Scientific	Cat# 11496015
High Five (BTI-TN-5B1-4) cells	Thermo Fisher Scientific	Cat# B85502
Experimental Models: Organisms/Strains
Schizosaccharomyces pombe	This paper	NCBI:txid4896
h- ade6-M210 leu1-32 ura4-D18	This paper	KS516
h- hphMX6:nmt81:3xFLAG-mzt1 ade6-M210 leu1-32 ura4-D18	This paper	KS7623
h- mto1(131-549)-mEcitrine:kanMX6 natMX6:Z:adh15:mCherry-Atb2 ade6-M210 ura4-D18 leu1-32	This paper	KS10055
h- hphMX6:nmt81:3xFLAG-mzt1 mto1(131-549)-mEcitrine:kanMX6 natMX6:Z:adh15:mCherry-Atb2 ade6-M210 ura4-D18 leu1-32	This paper	KS10059
Oligonucleotides
Primers for PCR, see Table S1	This paper	N/A
Recombinant DNA
pFL vector	Imre Berger Lab [38];	pKS1219
pUCDM vector	Imre Berger Lab [39];	pKS1251
pMK-RQ XhoI_L21_Gtb1_NheI (to generate untagged Gtb1)	GeneArt (this paper)	pKS1815
pMA BamHI_Alp4_TAA_PstI (to generate untagged Alp4)	GeneArt (this paper)	pKS1807
pMA BamHI_L21_Alp6_TAA_PstI (to generate untagged Alp6)	GeneArt (this paper)	pKS1812
pMK-RQ KasI_MBP_His_PstI (to generate C-terminal MBP-6xHis tags)	GeneArt (this paper)	pKS1811
pMK-RQ BamHI_Alp4_KasI (to generate tagged Alp4)	GeneArt (this paper)	pKS1814
pMK-RQ BamHI_L21_Alp6_KasI (to generate tagged Alp6)	GeneArt (this paper)	pKS1810
pMA-T Mzt1 (insect cells) (to generate Mzt1 codon-optimized for insect cell expression)	GeneArt (this paper)	pKS1813
pFL_Alp6-MBP_Gtb1	This paper	pKS1791
pUCDM_Alp4_Gtb1	This paper	pKS1788
γ-TuSC Alp6-MBP	This paper	pKS1794
pFL_Alp4_Gtb1	This paper	pKS1804
pFL_Mzt1-6xHis	This paper	pKS1801
pFL_6xHis-Mto1/2[bonsai]	This paper	pKS1225
pFL_GST-Mto1bonsai	This paper	pKS1548
pFL_6xHis-Mto2	This paper	pKS1258
pUCDM_Alp6_Gtb1	This paper	pKS1785
γ-TuSC Untagged	This paper	pKS1805
pFL_Alp4-MBP	This paper	pKS1795
pFL_Alp6-MBP	This paper	pKS1796
pFL_Strep-Mto1/2[bonsai]	This paper	pKS1799
p0GWA vector	Didier Busso Lab [40];	pKS537
pGGWA vector	Didier Busso Lab [40];	pKS538
pMA-T Mzt1_bac (to generate Mzt1 codon-optimized for bacterial expression)	GeneArt (this paper)	pKS1809
p0GWA Mzt1_6xHis	This paper	pKS1821
pGGWA GST_Mzt1_6xHis	This paper	pKS1822
pFA6a-hphMX6-P81nmt1-3FLAG	Addgene [41];	RRID:Addgene_19350; pKS1284
pFA6a-mEcitrine-KanMX6	Addgene [42];	RRID:Addgene_105150; pKS1330
Software and Algorithms
ImageJ (Fiji)	NIH	RRID:SCR_002285
GraphPad Prism 7 software	GraphPad	RRID:SCR_002798
S. pombe database (released in August, 2013)	PomBase	RRID:SCR_006586
Image Studio Lite	LI-COR	RRID:SCR_013715
Image Lab™	Bio-Rad	RRID:SCR_014210
Other
Bolt® 4-12% Bis-Tris Gel	Thermo Fisher Scientific	Cat# NW04125BOX; Cat# NW04127BOX; Cat# NW04122BOX; Cat# NW04120BOX
RunBlue™ 4-20% Teo-Tricine SDS Gels	Expedeon	Cat# NXG42012
ÄKTA chromatography system	GE Healthcare	N/A
ÄKTAmicro chromatography system	GE Healthcare	N/A
Viscotek SEC-MALS 20 detector	Malvern Instruments	N/A
Viscotek RI detector VE3580	Malvern Instruments	N/A
Amicon Ultra-4 Ultracel-3K centrifugal filter unit	Millipore	Cat# UFC800324
Amicon Ultra-15 Ultracel-3K centrifugal filter unit	Millipore	Cat# UFC900324
Half area 96 well plate	Greiner Bio-One Inc.	Cat# 675076
Coverslips 24x50 mm No. 1.5	VWR	Cat# 631-0147
Gradient Master 107ip	Biocomp Instruments	N/A
SpectraMax M5 multi-mode microplate reader	Molecular Devices	N/A
Superose 6 10/300	GE Healthcare	Cat# 17-5172-01
Superdex 75 10/300	GE Healthcare	Cat# 17-5174-01
Beckman Coulter Optima MAX Ultracentrifuge	Beckman Coulter	N/A
ChemiDoc™ Imager	Bio-Rad	Cat# 17001401
Odyssey CLx fluorescence imager	LI-COR	N/A
Airyscan confocal microscope	Zeiss	LSM880
Freezer/mill® LLC 6870	SPEX SamplePrep	N/A
400 mesh copper grid with carbon film support	TAAB	Cat# C169/100
Filter paper, grade 1	Whatman	Cat# 1001-090
JEM-1400 transmission electron microscope	JEOL	JEM-1400
OneView 4k x 4k CMOS detector	Gatan	N/A