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

mryner@uw.edu