Publications

The accessory Sec (SecA2/Y2) systems of streptococci and staphylococci are dedicated to the transport of large serine-rich repeat (SRR) glycoproteins to the bacterial cell surface. The means by which the glycosylated preproteins are selectively recognized by the accessory Sec system have not been fully characterized. In Streptococcus gordonii, the SRR glycoprotein GspB has a 90-residue amino-terminal signal sequence that is essential for transport by SecA2/Y2 but is not sufficient to mediate the transport of heterologous proteins by this specialized transporter. We now report that a preprotein must remain at least partially unfolded prior to transport by the accessory Sec system. In addition, a region of approximately 20 residues from the amino-terminal end of mature GspB (the accessory Sec transport or AST domain) is essential for SecA2/Y2-dependent transport. The replacement of several AST domain residues with glycine strongly interferes with export, which suggests that a helical conformation may be important. Analysis of GspB variants with alterations in the AST domain, in combination with the results with a SecY2 variant, indicates that the AST domain is essential both for targeting to the SecA2/Y2 translocase and for initiating translocation through the SecY2 channel. The combined results suggest a unique mechanism that ensures the transport of a single substrate by the SecA2/Y2 system.

RATIONALE

Induced pluripotent stem (iPS) cells can differentiate into multiple cell types, including cardiomyocytes and have tremendous potential for drug discovery and regenerative therapies. However, it is unknown how much variability exists between differentiated lineages from independent iPS cell lines and, specifically, how similar iPS cell-derived cardiomyocytes (iPS-CMs) are to embryonic stem (ES) cell-derived cardiomyocytes (ES-CMs).

OBJECTIVE

We investigated how much variability exists between differentiated lineages from independent iPS cell lines and how similar iPS-CMs are to ES-CMs.

METHODS AND RESULTS

We generated mouse iPS cells in which expression of NKX2-5, an early cardiac transcription factor, was marked by transgenic green fluorescent protein (GFP). Isolation of iPS- and ES-derived NKX2-5-GFP(+) cardiac progenitor pools, marked by identical reporters, revealed unexpectedly high similarity in genome-wide mRNA expression levels. Furthermore, the variability between cardiac progenitors derived from independent iPS lines was minimal. The NKX2-5-GFP(+) iPS cells formed cardiomyocytes by numerous induction protocols and could survive upon transplantation into the infarcted mouse heart without formation of teratomas.

CONCLUSIONS

Despite the line-to-line variability of gene expression in the undifferentiated state of ES and iPS cells, the variance narrows significantly in lineage-specific iPS-derived cardiac progenitors, and these progenitor cells can be isolated and used for transplantation without generation of unwanted cell types.