<13.03.23 >

INHA UNIVERSITY- 김준섭

▶Objective : P protein 이 Tyrosine 과 Tyrosinase 의 localization 에 영향을 미친다는 가설에 대한 논문을 알아보기

The pink-eyed dilution phenotype in mice arises from mutations in the p gene; in humans, analogous mutations in the P gene result in oculocuta-neous albinism type 2. Although the molecular mechanisms which underlie this phenotype remain obscure, it has been postulated that mutations in p result in defective tyrosine transport into murine melanosomes, resulting in hypopigmentation and diminished coat color. However, we previously reported no difference in melanosomal tyrosine transport in unpigmented, melanoblast-like pinkeyed dilution (pcp/pcp), and in pigmented (melan-a) murine melanocytes. In this study, we utilized melan-p1 cells, more differ-entiated pink-eyed dilution ( pcp/p25H) melanocytes which can be induced to produce melanin, to characterize the melanogenic lesion(s) more defini-tively. Uptake of [3H]tyrosine into melan-a melanosomes did not differ sig-nificantly from uptake into melanosomes derived from melan-p1 melanocytes, further arguing against its critical role as a tyrosine trans-porter. Pink-eyed dilution melanocytes incubated in high tyrosine concen-trations became extremely pigmented as they became confluent and se-creted large amounts of black material into the medium. Total cellular ty-rosinase activity in melan-p1 melanocytes was significantly higher than that in melan-a melanocytes (which are wild-type at the p locus), but the localization of tyrosinase to melanosomes was impaired in melan-p1 melanocytes compared to melan-a melanocytes. These results indicate that mechanisms other than deficient tyrosine transport are involved in the pink-eyed dilution phenotype and that this protein may serve a chap-erone-like or stabilizing function in melanocytes. © 1998 Academic Press

ABSTRACTS

ABSTRACTS

P protein tyrosine transport

wild-type : Melan-a (a/a, p/p)

P mutant : Melan-p1 (a/a, pcp/

p25H)

p gene oculocutaneous albinism type 2mutation

<Postulation>

<In this study>

p gene tyrosine transport X

cellular tyrosinase activity : melan-p1 > melan-a

melan-p1 : localization of tyrosinase to melanosomes was im-

paired.<mechanisms>

P protein may serve a chaperone-like or stabilizing function in melanocytes.

INTRODUCTION

tyro-sine~!

P protein 이 tyrosine 을 transport 할까요 ?

NO~!

melanosome

•carrier-mediated

process

•energy-requiring

•affected by a pH gradi-

ent

tyrosinase : melan-p1 cells > melan-a cells

melan-p1 : melanogenic enzyme to melanosomes was dramatically impaired

p protein plays an important role in localizing and/or stabilizing tyrosinase in the melanosome.

MATERIAL AND METHODS

Cell culture : wild-type : Melan-a (a/a, p/p) C57BL/6 mice

P mutant : Melan-p1 (a/a, pcp/p25H) pink eyed dilution

mice

Tyrosine transport assays : [3H-tyrosine]

Subcellular fractionation : tyrosine transport assay, tyrosinase ac-

tivity

Light microscopy

Enzyme assays and protein determinations : tyrosine hydroxy-

lase assay

Western immunoblotting analysis

RESULTS

1. Tyrosine Transport Studies

FIG. 1. [3H]Tyrosine up-take into melanosomes derived from melan-a or melan-p1 melanocytes.

darker bars : melan-a

lighter bars : melan-p1

melan a 와 melan-p 는 별 차이가 없다 .

tyrosine transport : 50uM 이 10uM 약 2 배

tyrosine transport X

RESULTS2. Tyrosine Induction of Melanin Production

FIG. 2. Melanin se-creted into media by melan-a or melan-p1 melanocytes.

(4) melan-p, 1.0 mM tyr.

(1) melan-a,

0.2 mM tyr

(2) melan-p, 0.2 mM tyr

(3) melan-a, 1.0 mM tyr

Melanin : melan-p > melan-a

tyrosine

tyrosine melan-p1

<melanin extracellular medium>

RESULTS

FIG. 3. Phenotypic appearance of melanocytes cultured at varying concentrations of tyrosine. Bar, 50 um.

2. Tyrosine Induction of Melanin Production

<tissue chamber slides>

melan-a : relatively stable

melan-p1 : tyrosinase den-

dritic

<phenotype>

RESULTS3. Subcellular Distribution of Tyrosinase

tyrosine hydroxlase assay tyrosinase activity

tyrosinase activity : melan-p1 > melan-a

melan-a : melanosome > cell extract (5 배 )

melan-p1 : melanosome < cell extract

RESULTS

FIG. 4. Tyrosinase in subcellular fractions of melan-a or melan-p1 melanocytes.

Ext : total extract of cellsMS : melanosomal frac-tion

imimmunoreactive tyrosinase present in cell extracts and in melanosome fractions

3. Subcellular Distribution of Tyrosinase

<Western immunoblot-ting>tyrosinase amount : melan-a > melan-p1

αPEP-7 antibodytyrosinase 의 carboxylic tailmelanosome 에 위치한 ty-rosinase 를 잘 인지

<conflict>

DISCUSSION

•tyrosinase•tyrosinase stability•melanogenic enzymatic func-tions•melanin production

p gene muta-tion

result

How-ever,

tyrosinase activity : melan-p1 > melan-a

Why?

•studies were performed on extracts of tissues

•melanocytes are a relatively small percentage of the total

population (1-2%)

•decreased numbers of mutant elanocytes are present.

p protein plays an important role in assembling and stabilizing a complex of melanosomal proteins which includes tyrosinase