Caenorhabditis elegans sperm membrane protein interactome

Abstract

The interaction and organization of proteins in the sperm membrane are important for all aspects of sperm function. We have determined the interactions between 12 known mutationally defined and cloned sperm membrane proteins in a model system for reproduction, the nematode Caenorhabditis elegans. Identification of the interactions between sperm membrane proteins will improve our understanding of and ability to characterize defects in sperm function. To identify interacting proteins, we conducted a split-ubiquitin membrane yeast two-hybrid analysis of gene products identified through genetic screens that are necessary for sperm function and predicted to encode transmembrane proteins. Our analysis revealed novel interactions between sperm membrane proteins known to have roles in spermatogenesis, spermiogenesis, and fertilization. For example, we found that a protein known to play a role in sperm function during fertilization, SPE-38 (a predicted four pass transmembrane protein), interacts with proteins necessary for spermiogenesis and spermatogenesis and could serve as a central organizing protein in the plasma membrane. These novel interaction pairings will provide the foundation for investigating previously unrealized membrane protein interactions during spermatogenesis, spermiogenesis, and sperm function during fertilization.

Figure 1.

Diagram of sperm development and function in C. elegans. During sperm development in C. elegans, primary spermatocytes divide into two secondary spermatocytes, which divide into two haploid spermatids each that are connected by a residual body [13, 14]. The membranous organelles (MOs) are Golgi-derived vesicles that are located near the plasma membrane of spermatids [13]. During spermiogenesis or post-meiotic sperm development, C. elegans sperm undergo a morphological change from a quiescent, round spermatid to an amoeboid sperm and gain motility, and the MOs fuse with the plasma membrane [13]. After spermiogenesis, the sperm are functional and can travel to the site of fertilization and fertilize the oocyte [13]. The approximate functional timing of the sperm genes that encode transmembrane proteins are indicated above the appropriate stage.

Figure 2.

Sample results from a MYTH system to assay membrane interactions between sperm proteins. A split-ubiquitin based membrane yeast two-hybrid (MYTH) system was employed to detect interactions between SPE proteins containing a transmembrane domain. (A) Genes necessary for sperm development and function were cloned into expression vectors based on their predicted membrane topology in order to generate a protein with split-ubiquitin fused to the cytosolic terminus of the protein. (B) Yeast expressing the indicated bait and prey vectors were plated at three different dilutions for each experiment (1×, 10×, 100×). Empty prey vectors (pR3-N and pPR3-STE, respectively) were used to determine the background interactions. The positive control expresses a fusion of the yeast ER protein Alg5 to the N-terminus of ubiquitin (Nub).

Figure 3.

Caenorhabditis elegans sperm membrane protein interactome. Network diagram generated from MYTH interaction data. The C. elegans sperm membrane interactome cluster is demarcated by a red outline. [blue nodes = genes necessary for spermiogenesis; orange nodes = genes necessary for spermatogenesis; green nodes = genes necessary for fertilization; circular nodes = proteins capable of homotypic interaction (self-looping node); square node = proteins not capable of homotypic interaction].

Figure 4.

Caenorhabditis elegans sperm membrane interactions during development. Sperm protein complex composition changes depending on the stage of sperm development. In spermatids, FER-1, SPE-4, SPE-10, SPE-38, and SPE-41/TRP-3 are localized to the MO (circled in red). The data presented here indicated that these proteins interact with SPE-19, SPE-29, and SPE-42 and form a sperm membrane complex in the MO of spermatids. Similar sperm membrane complex models are presented for MO, plasma membrane, and pseudopod localization by adding the interaction data presented here with the known protein localization. [red circle = protein localization based on published data; blue nodes = genes necessary for spermiogenesis; orange nodes = genes necessary for spermatogenesis; green nodes = genes necessary for fertilization; circular nodes = proteins capable of homotypic interaction (self-looping node); square node = proteins not capable of homotypic interaction].