Processing by the signal peptidase complex (SPC) is critical to the localization and function of secretory and membrane proteins which must enter the endoplasmic reticulum (ER) before they can be directed to their final destination. As proteins are transferred into the ER, the SPC cleaves the signal peptide sequence (SP), an N-terminal stretch of amino acids – usually 20–30 residues in length – that directs proteins to the ER 
. SPs possess a tripartite structure that includes a positively charged amino terminal domain, a 7–13 residue hydrophobic domain, and a hydrophilic domain that includes the cleavage site 
. While the SP sequence is not conserved, the properties associated with each domain are static and ensure that SP-bearing polypeptides are recognized by the cell and translocated into the ER 
Signal peptidases have been extensively studied in yeast and bacteria, yet little has been done to investigate their role in multicellular organisms. In S. cerevisiae
, four proteins, Sec11, Spc1p, Spc2p and Spc3p, comprise the SPC 
(). Sec11 and Spc3p are required for SPC catalytic function and cell viability. Temperature-sensitive sec11
mutants accumulate uncleaved SPC targets at non-permissive temperatures, indicating that both are required for SP cleavage 
. Spc1p and Spc2p do not have catalytic function and are dispensable for SPC cleavage activity and viability at normal growth temperatures. However, over-expression of Spc1p attenuates the sec11
temperature-sensitive phenotype 
, while depletion of Spc2p at high temperatures leads to the accumulation of uncleaved protein 
, suggesting that Spc1p and Spc2p contribute to SPC function in yeast although the mechanism has yet to be identified.
The SPC is conserved from yeast to humans.
In mammals, the SPC consists of five subunits: SPC18, SPC21, SPC22/23, SPC12 and SPC25 
. SPC18 and SPC21 have high identity to each other 
and are homologous to Sec11 
. SPC22/23 is homologous to Spc3p 
while SPC12 and SPC25 are homologous to Spc1p and Spc2p, respectively 
(). SPC18, SPC21 and SPC22/23 are single-pass transmembrane proteins, the bulk of which reside within the ER lumen. SPC12 and SPC25 are double-pass transmembrane proteins each containing a small lumenal domain, while the N- and C-termini of both are cytosolic 
. SPC18, SPC21, and SPC22/23 have catalytic function and the residues required for cleavage activity are localized to the ER lumen 
Four SPC homologs have been identified in Drosophila
: Spase18/21, Spase22/23, Spase12 and Spase25 (). Spase18/21 is homologous to yeast Sec11, as well as mammalian SPC18 and SPC21 
. ER vesicles (microsomes) purified from Drosophila
embryos and added to an in vitro
translation system results in cleavage of murine myeloma light-chain IgG, demonstrating that the Drosophila
SPC is functionally conserved 
Despite playing a key role in protein sorting, in vivo
studies of SPC function in metazoans have not been reported. We have used the Drosophila
eye as a model system to investigate the role of spase12
and the SPC in a higher eukaryote. The eye originates from a developmental structure called the eye imaginal disc, an epithelial monolayer of cells that begin to differentiate during the third instar larval stage. The eye continues to develop through larval and pupal stages into a highly organized array comprised of approximately 800 unit eyes (ommatidia). Each ommatidium contains eight photoreceptor cells and four cone cells enclosed by two primary, six secondary, and three tertiary pigment cells, as well as three interommatidial bristles (IOBs) 
. The genetic approaches available in Drosophila
, coupled with the well characterized development and structure of the eye, make it an ideal model for developmental studies.
In this report, we characterize spase12 loss-of-function (LOF) phenotypes in the Drosophila eye through clonal analysis. Our findings show that spase12 mutants are embryonic lethal, while spase12 LOF clones result in developmental defects in all tissues tested. Specifically, spase12 LOF in the Drosophila eye leads to errors in cell differentiation. Together, these data indicate that spase12 is required for viability, development, and differentiation.