714 Preferential localization of BCL-2 and its homologous proteins to the inner mitochondrial membrane

S83 CLONING AND ANALYSIS OF KAP3: A NOVEL KINESIN SUPERFAMILY ASSOCIATED PROTEIN OF THE HETERODIMERIC KIF3A/B . HIROTO YAMAZAK I, TAKAO NAKATA, YASUSHI OKAD and NOBUTAKA HIROKAWA, Department of Anatomy and Cell Biology, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan 712

We previously reported that KIF3A and KIF3B form a heterodimer that functions as a microtubule-based fast anterograde translocator of membranous organelles. We have also shown that this KlF3A/B forms a complex with other associated polypeptides, named kinesin superfamily associated protein 3 (KAP3). In the present study, we purified KAP3 protein by immunoprecipitation using anti-KIFSB antibody from mouse testis. Microsequencing was carried out and we cloned the full-length KAP3 cDNA from a mouse brain cDNA library. Two isoforms of KAP3 exist (KAP3A, 793 a.a.: KAP3B, 772 a.a.), generated by alternative splicing in the carboxyl-terminus region. Their amino acid sequences have no homology with those of any other known proteins and prediction of their secondary structure indicated that almost the entire KAP3 molecule is a-helical. We produced recombinant KAP3 and KIF3A/B using a baculovirus-Sf9 expression system. A reconstruction study in Sf9 cells revealed that KAP3 is a globular protein that binds to the tail domain of KIF3A/B. The immunolocalization pattern of KAP3 was similar to that of KIF3A/3B in nerve cells. In addition, we found that KAP3 does not affect the motor activity of KIF3A/B. KAP3 was associated with a membrane-bound form of KIFXVB in a fractional immunoprecipitation experiment, and since the KIF3 complex was found to bind to membranous organelles in an EM study, KAP3 may regulate membrane binding of the KlF3 complex.

Synapmin I deficiency resulta in thm structural change in the prasynaptic terminals in the murine PM-VOU~ system. Yoauke Takeil*48 Akihiro Haradal,z, Sen Takodal, Katsunori Kobayashis, Sumio Toradalna, Tbtsuo sodas, Tomoyuki Takahashia, and Uobutaka Hirokawalez IDepartment of Anatomy and Cell Biology, Faculty of Medicine, University of Tokyo; 2Institute Brain Research, Department of Neuroanatomy, Faculty of Medicine, University of Tokyo; 'Institute of Brain Research, Department of Neurophysiology, Faculty of Medicine, University of Tokyo; 4Department of Neurobiology and Neuropathology, Faculty of Pharmaceutical Sciences, University Tokyo: SDepartment of Cell Biology, Cancer Institute, JAPAN. 713

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of

Synapsin I is one of the major synaptic vesicle-associated proteins. Previous experiments implicated its crucial role in synaptogenesis and transmitter release. To better define the role of synapsin I in viva, we used gene targeting to disrupt the murine synapsin I gene. Mutant mice lacking synapsin I did not have gross anatomical abnormalities. However, when we examined the presynaptic structure of the hippocampal CA3 field in detail, we found that the sizes of mossy fiber giant terminals were significantly smaller, the number of synaptic vesicles became reduced, and the presynaptic structures altered, although the mossy fiber long-term potentiation (LTP) remained intact. These results suggest significant contribution of synapsin I to the formation and maintenance of the presynaptic structure.

PREFERENTIAL

714

LOCALIZATION

MEMBRANE. Osaka,

OF BCL-2

GOTOW,

AND

YASUO

ITS HOMOLOGOUS

UCHIYAMA,

PROTEINS

TO THE INNER

Dept. of Cell Biol. and Anat.

I, Osaka

MITOCHONDRIAL

Univ. Med. Sch., Suita,

565, Japan.

Bcl-2 they

TAKAHIRO

and its homologous

regulate

necessary

to accurately

the human

bcl-2

subcellular

fractionation

and monoclonal proteins

define and

bcalizations

almost

the identical

enriched

in the mitochondrial

sections

in which

there

suggest

role in the regulation

human

fraction.

was hardly

is controversial.

membrane

protein,

of apoptosis

including

in concert

the cryothin

and rat Bci-2, BclxL which

also very

similar

supernatant

association

labeling proteins

w

nuclear

in other

We found

gold

the PC12

membrane except

membranes

into the inner

B&XL

with various

revealed

mitochondrial

with

polycbnal that these

by vital staining that Bd-2

was distinctly

shown

that of the endoplasmic membrane

how

and Bax using

laser microscopy

cells demonstrated

of inner mitochondrial

functions.

technique

unknown

first of all, it is

cells transfected

proteins,

with confocal

to that of mitochondria

from

remains of Bcl-2,

We used PC12

organelle

are integrated

with the inner membrane

especially

and its related

section-colloidal or Bax.

but it totally

proteins,

localization

The preferential

that Bcl-2 and its related

(apoptosis),

of these

since its subcelluar

of the post

any significant

cell dath

mechanism

of Bcl-2, an integral , mouse

distribution

fractionation

of programmed

the precise

immunocytochemistry against

Subcelluar

as regulators

To clarify

their localization

antibodies

123.

We therefore

function

of cell death.

gene to examine

showed

rhodamine

proteins

the process

of

was mOSt in cryothin reticulum.

and play an essential