Бесплатный автореферат и диссертация по биологии на тему

Генетический анализ роли гликозилфосфатидилинозита в биогенезе клеточной стенки и сворачивании секретируемых белков дрожжей Saccharomyces Cerevisiae

ВАК РФ 03.00.15, Генетика

Содержание диссертации, кандидата биологических наук, Фоминов, Глеб Вадимович

СПИСОК СОКРАЩЕНИЙ.

ВВЕДЕНИЕ.

ОБЗОР ЛИТЕРАТУРЫ.

1. Введение: структура и функции клеточной стенки.

2. Синтез глюканов клеточной стенки.

2.1. Синтез Р-1,3-глюканов клеточной стенки.

2.1.1. Р-1,3-глюкансинтетаза.

2.1.2. Ген НШ1.

2.1.3. Семейство генов, родственных

2.1.4. Семейство генов, родственны\BGL2.

2.2. Синтез Р-1,6-глюканов.

2.2.1. Ген КЯЕ5.

2.2.2. Ген ВЮ1.

2.2.3. С1УН41/СЬ81, 1ЮТ2ЮЬ82 и некоторые другие гены, кодирующие белки, участвующие в синтезе А^-связанных гликанов и сворачивании секретеру емых белков.

2.2.4. Гомологичные гены КЯЕ6 и БЮЛ.

2.2.5. Ген КЯЕП/тЯбЗ.

2.2.6. Ген КЯЕ1.

2.2.7. Гены КЯЕ9 и КЫН1.

3. Синтез хитина клеточной стенки.

3.1. Хитинсинтетаза 1.

3.2. Хитинсинтетаза II.

3.3. Хитинсинтетаза Ш.

4. Синтез ^-связанных гликанов секретируемых белков.

4.1. Формирование коровой части.

4.2. Реакции, протекающие в комплексе Гольджи: формирование «внешних цепей» или «зрелого кора».

5. Синтез О-связанных гликанов секретируемых белков.

6. Формирование гликозилфосфатидилинозитного (GPI) якоря.

6.1. Структура GPI якоря.

6.2. Формирование GPI якоря и его присоединение к белку.

6.3. События, следующие за присоединением GPI якоря к белку.

6.3.1. Деацилирование GPI.

6.3.2. Модификация липидной составляющей GPI якоря.

6.3.3. Добавление пятого остатка маннозы.

6.3.4. Встраивание некоторых белков, несших GPI якорь, в КС.

7. Сворачивание секретируемых белков.

7.1. Связанная с ЭР деградация неправильно свернутых белков (ERAD, endoplasmic reticulum associated degradation).

7.2. Ответ клетки на накопление в ЭР неправильно свернутых белков (UPR, unfolded protein response).

7.3. Роль ионов кальция в сворачивании секретируемых белков.

Введение Диссертация по биологии, на тему "Генетический анализ роли гликозилфосфатидилинозита в биогенезе клеточной стенки и сворачивании секретируемых белков дрожжей Saccharomyces Cerevisiae"

В настоящее время дрожжи ЗассИаготусез сегеугзгае являются одним из наиболее популярных объектов молекулярных биологов. Это связано, во-первых, с тем, что при изучении их клеток легко применимы методы как классической, так и молекулярной генетики. Во-вторых, на сегодняшний день расшифрована последовательность всех генов дрожжей, что существенно упрощает работу. В-третьих, дрожжи обладают очень высокой скоростью роста, что ускоряет проведение экспериментов. Кроме того, для выращивания дрожжей можно использовать относительно простые по составу и недорогие среды. Стоит также отметить, что, поскольку дрожжи являются представителями одноклеточных эукариот, многие данные, полученные при использовании этого модельного объекта, могут быть экстраполированы на клетки высших эукариот.

Клетки всех эукариот и некоторых архебактерий способны синтезировать гликозилфосфатидилинозит (СР1). При этом структура СР1 якоря, связывающего некоторые белки с мембраной (см. Обзор литературы), имеет много общих черт у дрожжей и млекопитающих, что, как было упомянуто выше, позволяет использовать данные, полученные при работе с клетками дрожжей, при изучении клеток высших эукариот. В тоже время, выявление особенностей синтеза СР1, свойственных только клеткам дрожжей, делает возможным разработку антигрибковых лекарственных препаратов, мишенями которых являются реакции, специфичные для клеток грибов.

Данная работа посвящена изучению взаимосвязи формирования вР1 с другими процессами, протекающими в клетках дрожжей, такими, как биогенез клеточной стенки и сворачивание секретируемых белков. Ранее в нашей лаборатории был получен штамм, несущий мутацию в гене МСВ4 (Раск^Бег й а1., 1999). Эта мутация приводит к нарушению синтеза вР1 (ЬпЬоГ е1 а1., 2004), однако, кроме этого она обладает фенотипическими проявлениями, не связанными очевидным образом с нарушением синтеза гликозилфосфатидилинозита, такими, например, как чувствительность к кофеину и пониженному содержанию ионов кальция в среде (Packeiser et al., 1999). Это позволило предположить наличие у белка Mcd4p дополнительных функций. Таким образом, целью данной работы было прояснение физиологической роли белка Mcd4p, в связи с чем представлялось интересным выявить гены, функционально связанные с геном MCD4, и выяснить причины возникновения у мутации ssu21 в гене MCD4 фенотипических проявлений, не связанных явным образом с нарушением синтеза GPI.

ОБЗОР ЛИТЕРАТУРЫ

Заключение Диссертация по теме "Генетика", Фоминов, Глеб Вадимович

ВЫВОДЫ

1. Мутантная аллель ssu21 гена MCD4 содержит 2 мутации. Первая из них приводит к замене Argl05->Cys в кодируемом белке, вторая - к сдвигу рамки считывания, в результате чего должен синтезироваться укороченный с С-конца белок.

2. Получены 6 мутаций, летальных или полулетальных в комбинации с мутацией ssu21. Некоторые из этих мутаций приводят к нарушению формирования клеточной стеики и/или морфологии клеток.

3. Идентифицированы гены MNN9 и GWT1, комплементирующие фенотипические проявления 2-х из 6-ти полученных мутаций.

4. Показано, что белок Gwtlp локализован в эндоплазматическом ретикулуме.

5. Клонированы гены PSD1, НАС1, UBI4 и SK01, способные в мультикопийном состоянии супрессировать чувствительность мутанта ssu21 к кофеину.

6. Полученные результаты позволяют предположить, что причиной чувствительности мутанта ssu21 к кофеину и пониженному содержанию ионов кальция в среде является накопление в секреторных путях неправильно свернутых белков.

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284. Выражаю благодарность моему научному руководителю М.Д. Тер-Аванесяну за терпеливое руководство и помощь в работе.