FIG. 1.

The diverse chemical environments within cellular organelles pose undesirable consequences for fluorescent proteins (FPs). (A) The organelles of the eukaryotic secretory pathway maintain distinct luminal pH values. FPs targeted to the secretory or endosomal pathways will encounter pH values that range from 7.2 to 5.7 or 6.5 to 4.5, respectively. Therefore, it is important to consider the pKa values of FPs in these environments. (B) Secretory proteins with cysteine(s) and/or glycosylation consensus sequences are modified as they traverse through the secretory pathway. In the endoplasmic reticulum (ER), N-linked sugars are added covalently, and as proteins within the secretory pathway continue to the Golgi complex, N-linked sugars are modified and potential O-linked glycosylation can be added as well. Cysteines have the potential to form disulfide bonds, primarily within the ER, although the entire secretory pathway is effectively oxidizing. (C) Eukaryotic (secretory pathway and the intermembrane space of chloroplasts and mitochondria) and prokaryotic (gram-negative bacteria periplasm) cellular environments with oxidizing environments, depicted with gray coloring.