Mandy_C2B_bindingnucleicacidcontamination

Mandy_C2B_bindingnucleicacidcontamination - Biochemistry...

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Accelerated Publications The C 2 B Domain of Synaptotagmin I Is a Ca 2 + -Binding Module ² Josep Ubach, ‡,§ Ye Lao, | , ,# Imma Fernandez, ‡,§ Demet Arac, ‡,§ Thomas C. Su ¨dhof, | , ,# and Josep Rizo* ,‡,§ Departments of Biochemistry, Pharmacology, and Molecular Genetics, Center for Basic Neuroscience, and Howard Hughes Medical Institute, Uni V ersity of Texas Southwestern Medical Center, 5323 Harry Hines Boule V ard, Dallas, Texas 75390 Recei V ed February 16, 2001; Re V ised Manuscript Recei V ed April 2, 2001 ABSTRACT : Synaptotagmin I is a synaptic vesicle protein that contains two C 2 domains and acts as a Ca 2 + sensor in neurotransmitter release. The Ca 2 + -binding properties of the synaptotagmin I C 2 A domain have been well characterized, but those of the C 2 B domain are unclear. The C 2 B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca 2 + -dependent manner, leading to an attractive model whereby Ca 2 + -dependent multimerization of synaptotagmin I via the C 2 B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca 2 + -dependent C 2 B domain dimerization, as well as in analyses of other C 2 B domain interactions. To shed light on these issues, the C 2 B domain has now been studied using biophysical techniques. The recombinant C 2 B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C 2 B domain interactions, including Ca 2 + -dependent dimerization. NMR experiments show that the pure recombinant C 2 B domain binds Ca 2 + directly but does not dimerize upon Ca 2 + binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C 2 A and C 2 B domains, participates in a high molecular weight complex as a function of Ca 2 + . These results show that the recombinant C 2 B domain of synaptotagmin I is a monomeric, autonomously folded Ca 2 + -binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C 2 B domains or requires a posttranslational modification. Synaptotagmins constitute a large family of neuronal membrane proteins ( 1 , 2 ). Synaptotagmin I, the first member identified in this family, is a synaptic vesicle protein that is essential for fast Ca 2 + -triggered neurotransmitter release ( 3 ), most likely acting as a Ca 2 + sensor in this process ( 4 , 5 ). Synaptotagmin I contains an N-terminal intravesicular se- quence, a transmembrane region, a linker sequence, and two C-terminal C 2 domains referred to as the C 2 A and C 2 B domains (Figure 1A). The synaptotagmin I C 2 A domain has a ± -sandwich structure and binds multiple Ca 2 + ions via a motif that includes five conserved aspartate residues at the
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Mandy_C2B_bindingnucleicacidcontamination - Biochemistry...

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