Characterizing the abalone sperm proteome
&the evolution of a newly discovered
sperm proteinMelody Rynerson Palmer, Willie Swanson
University of WashingtonDepartment of Genome Sciences
SMBE 2010
Reproductive Isolation
2
Post zygotic: hybrid sterility, inviability
Pre-zygotic: pre-mating: behavioral, temporal, spatialpost-mating: molecular incompatibility
Quantifiable molecular incompatibility: abalone sperm lysin activity
Modified from Swanson & Vacquier 1997
homospecificheterospecific
Lysi
n A
ctiv
ity
μg Purified Lysin
Species specific fertilization
WhiteGreen & Pink
Threaded
Red
Black
Flat
Pinto
Diverged < 18 MYA
Overlapping ranges & spawning times
Discus
Abalone Fertilization
Acrosome Reaction
Fusion of sperm and egg membranes
Vitellin Envelope (VE)Acrosome
Lysis of VE
Entry into perivitelline space
Adhesion to Vitelline Envelope (VE)
LysinVERL
sp18 VEZP14
Egg Cytoplasm
sp18
lysin16K
Ladd
er
Under positive selection
Genomic & Proteomic approaches in a non-model organism (Red Abalone)
1. Testis tissue transcriptomics- cDNA clone library- In progress: RNA-seq – high
throughput Illumina sequencing (Shendure Lab)
2. Sperm proteomics - mass spectrometry (MacCoss Lab)
Gonad
What other proteins can we find with proteomics?
sp18: sperm-egg fusion
lysin: dissolves VE
S ARM
16K
Sperm mass spectrometry
proteins peptides chromatographicseparation
MS/MS
precursor m/z = 690.2
Protein identification
RNA-seq and proteomics workflow
Testis Extracted mRNA
Sequenced 2000 cDNA
clones
1300 unique ESTs
Sperm Mass spectrometry
Search against EST
database Identified 90 sperm proteins
What other proteins are abundant in sperm?
24
20.1
14.2
6.5
kDa
USP144
10
• No match to known proteins
Mass Spectrometry Relative Protein Abundance
Closely related species show huge divergence in USP144
Red 1Red 2
Green 1Green 2
Discus
Black
Pinto 1Pinto 2Pinto 3
Signal Sequence
pI = 4.14, mature protein molecular weight ~6kD, charge ~ -11
Red 1 & Green 1 dN/dS = 2.3
Average dN/dS
lysin: 1.5 *sp18: 1.9 #
* Yang et al. 2000
# Vaquier et al. 1997
Mature Protein
Summary
• New technologies can be successfully applied in non-model organisms
• USP144 is abundant and rapidly evolving– What is its function?– What does it interact with?– Does it contribute to reproductive isolation?– What is its evolutionary history?
Purified USP144
5037
2520
15
10
75kDa
USP144
Acknowledgements