A Cell-Specific Transgenic Approach inXenopusReveals the Importance of a Functional p24 System for a Secretory Cell

Abstract

The p24␣, -␤, -␥, and -␦ proteins are major multimeric constituents of cycling endoplasmic reticulum-Golgi transport vesicles and are thought to be involved in protein transport through the early secretory pathway. In this study, we targeted transgene overexpression of p24␦ 2 specifically to the Xenopus intermediate pituitary melanotrope cell that is involved in background adaptation of the animal and produces high levels of its major secretory cargo proopiomelanocortin (POMC). The transgene product effectively displaced the endogenous p24 proteins, resulting in a melanotrope cell p24 system that consisted predominantly of the transgene p24␦ 2 protein. Despite the severely distorted p24 machinery, the subcellular structures as well as the level of POMC synthesis were normal in these cells. However, the number and pigment content of skin melanophores were reduced, impairing the ability of the transgenic animal to fully adapt to a black background. This physiological effect was likely caused by the affected profile of POMC-derived peptides observed in the transgenic melanotrope cells. Together, our results suggest that in the early secretory pathway an intact p24 system is essential for efficient secretory cargo transport or for supplying cargo carriers with the correct protein machinery to allow proper secretory protein processing. INTRODUCTIONTransport of cargo proteins through the early secretory pathway involves cargo selection, transport vesicle formation, quality control to recycle misfolded cargo, and cycling of the COPI-and COPII-coated vesicles between the endoplasmic reticulum (ER) and Golgi (Barlowe, 2000). One of the major constituents of the transport vesicles is the p24 family of type I transmembrane proteins that can be classified into four main subfamilies, designated p24␣, -␤, -␥, and -␦ (Schimmö ller et al., 1995;Stamnes et al., 1995;Sohn et al., 1996;Nickel et al., 1997;Dominguez et al., 1998). The p24 proteins share a number of structural characteristics, such as a relatively large lumenal putative cargo-binding domain, a coiled-coil region thought to be involved in the formation of multimeric p24 complexes, a transmembrane region, and a short cytoplasmic tail containing COPI-and COPII-binding motifs that are used for p24 traveling from the ER to the Golgi and back (for review, see Kaiser, 2000). In yeast and mammalian cells, p24 proteins form functional heterotetrameric complexes containing one representative of each subfamily, whereby the composition of the complex may differ in various cell types (Dominguez et al., 1998;Fü llekrug et al., 1999;Marzioch et al., 1999;Ciufo and Boyd, 2000;Emery et al., 2000;Belden and Barlowe, 2001). Furthermore, the stability of the p24 members seems to be compromised when cells are deficient in the expression of a single p24 protein (Marzioch et al., 1999;Denzel et al., 2000). Recent evidence suggests a complex and dynamic p24 system of mostly monomers and homo-/heterodimers and that the degree of oligomerization constantly alters and largely depends on the s...

The trafficking protein Tmed2/p24β1 is required for morphogenesis of the mouse embryo and placenta

“…A large number of studies have shown that the expression of individual TMED proteins is interdependent (Bouw et al, 2004; Denzel et al, 2000; Jenne et al, 2002; Luo et al, 2007; Marzioch et al, 1999; Muniz et al, 2000). Our own studies provide further support for this interdependency; however, the significance and requirement for this remain unclear.…”

“…Members of the TMED/p24 family fall into four subfamilies based on shared protein identity: α, β, δ and γ. Although these subfamilies are conserved in all animals and fungi, species-specific duplications and/or losses have resulted in varying numbers of genes in each TMED/p24 subfamily (Bouw et al, 2004; Carney and Bowen, 2004; Dominguez et al, 1998; Strating and Martens, 2009; Strating et al, 2009). Ten Tmed/p24 genes are present in mammals: five in the γ subfamily, Tmed1/p24γ 1 , Tmed3/p24γ 4 , Tmed5/p24γ 2 , Tmed6/p24γ 5 , and Tmed7/p24γ 3 ; three in the α subfamily, Tmed4/p24α 3 , Tmed9p24α 2 and Tmed11/p24α 1 ; one in the δ subfamily Tmed10/p24δ 1 ; and one in the β subfamily, Tmed2/p24β 1 (Strating et al, 2009).…”

LMAN1 and MCFD2 Form a Cargo Receptor Complex and Interact withCoagulation Factor VIII in the Early SecretoryPathway

“…Deletion of all p24 proteins in yeast produces only a mild secretory defect in a limited number of proteins (7). However, specific p24 genes may have diverse functions in other organisms (32)(33)(34). Interestingly, clear LMAN1 orthologs exist in invertebrates prior to the appearance of blood coagulation system components (35), including FV and FVIII, suggesting that this transport pathway may be required for a much broader array of protein cargo.…”

Isoform-selective Oligomer Formation of Saccharomyces cerevisiae p24 Family Proteins

“…p24 family proteins are evolutionarily conserved transmembrane proteins of ϳ24 kDa that have been characterized as cargo receptors for mammalian and yeast glycosylphosphatidylinositol-anchored proteins (24 -27), Drosophila Wingless protein (28,29), Xenopus pro-opiomelanocortin (30), and others (31,32). p24 proteins are single transmembrane proteins with a short C-terminal cytoplasmic tail of 10 -20 residues.…”