Title

The alpha3T gene of Drosophila melanogaster encodes a spermatogenesis-specific proteasome subunit

Date of Award

2001

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

John M. Belote

Keywords

Drosophila melanogaster, alpha3T, Proteasome subunit, Spermatogenesis

Subject Categories

Biochemistry, Biophysics, and Structural Biology | Life Sciences | Molecular Biology

Abstract

The proteasome is responsible for degradation of substrates of the ubiquitin pathway. 20S proteasomes are cylindrical particles with subunits arranged in a stack of four heptameric rings. The outer rings are composed of a subunits, and the inner rings are composed of β subunits. One major question in proteasome research concerns the extent and nature of proteasome structural heterogeneity. To investigate this topic, a molecular and genetic study of Drosophila proteasomes has been undertaken, with a goal of identifying proteasome subunit isoforms exhibiting developmental or cell-type specificity. Previous work led to the isolation of two proteasome subunit genes, encoding α4 isoforms that exhibit testis-specific expression (Yuan, et al., 1996). Here, I report the identification and characterization of a gene, α 3T , encoding a testis-specific isoform of another 20S proteasome subunit, α3.

The α 3T and α3 genes are unlinked, and they encode proteins that are 58% identical. Northern blot analyses reveal that α3 is expressed throughout development in both sexes, while α 3T is expressed primarily during the pupal and adult stages, and only in males. The tissue-specific expression pattern was examined in transgenic flies expressing GFP fusion proteins, revealing that the testis-specific α 3T -GFP only becomes prominent during and after meiosis, accumulates in the nucleus at later stages of spermatogenesis and is present in the head of mature, motile sperm. This pattern is in contrast to that of α3-GFP, which is expressed in all tissues examined. In testes, α3-GFP is prominently expressed in early stages of spermatogenesis in the cytosol and nucleus, but it fades from the elongating spermatid nuclei as the α 3T subunit accumulates. α 3T can functionally replace its yeast ortholog and, thus, encodes a functional proteasome subunit. Ectopic constitutive expression of α 3T in flies causes dominant pupal lethality, while expressing the α3 gene in the same way has no detectable consequence, suggesting that the α 3T subunit is functionally distinct from α3. In an attempt to silence α 3T gene expression, an inverted repeat of a portion of the α 3T gene (α 3T-IR ) was expressed in flies using the UAS/GAL4 system. The expressed α 3T-IR caused a dominant pupal lethality, instead of the anticipated male sterility phenotype.

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