A potential role for the clathrin adaptor GGA in Drosophila spermatogenesis

Jennifer Hirst,Jenny Carmichael

doi:10.1186/1471-2121-12-22

Abstract

BackgroundGGAs (Golgi-localised, γ-ear containing, ADP ribosylation factor-binding) are a family of clathrin adaptors that sort a number of biologically important transmembrane proteins into clathrin-coated vesicles. Knockout and knockdown studies to determine GGA function are confounded by the fact that there are 3 GGA genes in mammalian cells. Thus Drosophila melanogaster is a useful model system to study tissue expression profiles and knockdown phenotypes as there is a single GGA ortholog.ResultsHere we have quantified protein expression in Drosophila and show that there is >3-fold higher expression of GGA in male flies relative to female flies. In female flies the majority of GGA expression is in the head. In male flies GGA is not only expressed at high levels in the head but there is a gender specific increased expression which is due to the abundant expression of GGA in the testes. Using a highly specific antibody we have localised endogenous GGA protein in testes squashes, and visualised it in somatic and germ line cells. We show that GGA is expressed during multiple stages of sperm development, and co-stains with a marker of the trans-Golgi Network. This is most striking at the acroblast of early spermatids. In spite of the high expression of GGA in testes, knocking down its expression by >95% using transgenic RNAi fly lines did not affect male fertility. Therefore spermatogenesis in the male flies appears to progress normally with <5% GGA, most likely because alternative adaptors may be able to substitute partially or completely for the function of GGA. We also identify 'cueball' as a novel cargo for GGA, and mutants of cueball have been shown to have a male sterility phenotype.ConclusionIn Drosophila we have uncovered a potential role for GGA in the testes of male flies. The gender specific higher expression of GGA, its specific enrichment in testes and its localisation to developing spermatocytes and at the acroblast of spermatids supports a role for GGA function in Drosophila spermatogenesis, even though spermatogenesis still occurs when GGA expression is depleted to <5% of control.

Highlights

GGAs (Golgi-localised, g-ear containing, ADP ribosylation factor-binding) are a family of clathrin adaptors that sort a number of biologically important transmembrane proteins into clathrin-coated vesicles
The function of GGAs has been elucidated largely by knockout studies in yeast [2,3,4], and RNAi knockdowns in mammalian [5,6] and Drosophila tissue culture cells [6,7]. From these studies and others it is clear that GGAs function to sort a number of proteins for incorporation into clathrin-coated vesicles, including sortilin, cation-independent mannose 6-phosphate receptor (CIMPR), cation-dependent mannose 6-phosphate receptor, sorLA, LDL receptor-related proteins (LRP), bamyloid cleavage enzyme (BACE1), insulin-responsive aminopeptidase and stabilin-1 [[6], and references therein]
The only known GGA cargo in Drosophila is lysosome enzyme receptor protein (LERP), which is the functional equivalent of CIMPR, and like CIMPR contains both GGA and AP-1 clathrin adaptor sorting motifs [9]

Summary

Introduction

GGAs (Golgi-localised, g-ear containing, ADP ribosylation factor-binding) are a family of clathrin adaptors that sort a number of biologically important transmembrane proteins into clathrin-coated vesicles. The function of GGAs has been elucidated largely by knockout studies in yeast [2,3,4], and RNAi knockdowns in mammalian [5,6] and Drosophila tissue culture cells [6,7]. From these studies and others it is clear that GGAs function to sort a number of proteins for incorporation into clathrin-coated vesicles, including sortilin, cation-independent mannose 6-phosphate receptor (CIMPR), cation-dependent mannose 6-phosphate receptor, sorLA, LDL receptor-related proteins (LRP), bamyloid cleavage enzyme (BACE1), insulin-responsive aminopeptidase and stabilin-1 [[6], and references therein]. The only known GGA cargo in Drosophila is lysosome enzyme receptor protein (LERP), which is the functional equivalent of CIMPR, and like CIMPR contains both GGA and AP-1 clathrin adaptor sorting motifs [9]

Methods

Results

Discussion

Conclusion