Protein synthesis in synaptosomes: a proteomics analysis.
A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre-synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [(35)S] methionine and applied two-dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two-dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver-stained gels that were run in parallel. In-gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear-encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre-synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.
Jiménez
C R
CR
Graduate School of Neuroscience Amsterdam, Research Institute Neurosciences, Faculty of Biology, Vrije Universiteit Amsterdam, the Netherlands.
Eyman
M
M
Lavina
Z Scotto
ZS
Gioio
A
A
Li
K W
KW
van der Schors
R C
RC
Geraerts
W P M
WP
Giuditta
A
A
Kaplan
B B
BB
van Minnen
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D002490:Central Nervous System / Q000737:chemistry / Q000378:metabolism; D049832:Decapodiformes; D015180:Electrophoresis, Gel, Two-Dimensional; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D017403:In Situ Hybridization; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010786:Photoreceptor Cells / Q000378:metabolism; D017729:Presynaptic Terminals / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D020543:Proteome / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D020539:Sequence Analysis, Protein; D013574:Synaptosomes / Q000737:chemistry / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; D011506:Proteins; D020543:Proteome; D012333:RNA, Messenger