NOVEL KINESIN RELATED MOTOR PROTEIN

  • Scholey, Jonathan M (PI)

Project: Research project

Project Details

Description

Kinesin and kinesin-related proteins (KRPs) are microtubule (MT)-
based motor proteins that transport intracellular particles along
MTs, and thus play important roles in organelle placement, vesicle
trafficking, meiosis and mitosis. The aim of the work proposed here
is to characterize a novel trimeric kinesin related protein,
hereafter referred to as the KRP, from sea urchin (SU) eggs and
Drosophila embryos. The purified KRP, which is the first to be
isolated from its native cell-type, consists of two polypeptides
(Mr 85kd and 95kd) that have a kinesin motor domain complexed with
a 115kd accessory subunit. The KRP displays nucleotide sensitive MT
binding and bundling activity, and generates force for MT movement
in a motility assay. We propose a detailed analysis of the
molecular architecture of the trimeric KRP, its "in vitro" motor
activities and its biological functions, focusing mainly on the SU
egg KRP, although our analysis of the accessory polypeptide will
also involve the Drosophila KRP (whose purification protocol needs
to be completed). We will use monoclonal antibodies against the SU
motor subunits, and against the SU and fly accessory subunits, to
identify cDNAS encoding the corresponding polypeptides. These
probes will be used to learn the sequences and structural
relationships between the subunits in the native SU KRP. In
addition, the results of MT binding, ATPase and motility assays on
bacterially-expressed motor polypeptides and the native complex
will be compared, to illuminate the functional significance of
being components of a trimeric complex. We will probe the
biological functions of the complex using mAbs to the 85kd, 95kd
and 115kd subunits of the SU KRP for immunolocalization and
microinjection experiments on dividing sea urchin eggs and
blastomeres. Finally, we will study the function of the fly 115kd
homologue using antibodies for cytologicaI studies and by
generating mutants that are defective in 115kd function. Thus we
will test our hypothesis that, in mitotic cells, the 115kd subunit
may regulate or target the motor polypeptides to the site of force-
generation, where the KRP drives relative sliding between MTs, for
example during assembly or elongation of the mitotic spindle.
StatusFinished
Effective start/end date5/1/943/31/11

Funding

  • National Institutes of Health: $309,841.00
  • National Institutes of Health: $281,622.00
  • National Institutes of Health
  • National Institutes of Health: $286,111.00
  • National Institutes of Health: $295,009.00
  • National Institutes of Health: $15,242.00
  • National Institutes of Health: $255,897.00
  • National Institutes of Health: $280,216.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $295,048.00
  • National Institutes of Health: $285,289.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $248,528.00
  • National Institutes of Health: $286,932.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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