s-cyclophilin is retained intracellularly via a unique COOH-terminal sequence and colocalizes with the calcium storage protein calreticulin.
Cyclophilins (cyclosporin A-binding proteins) are conserved, ubiquitous, and abundant proteins that accelerate the isomerization of XaaPro peptide bonds and the refolding of proteins in vitro. s-Cyclophilin is a member of the cyclophilin family with unique NH2- and COOH-terminal extensions, and with a signal sequence. We now report that s-cyclophilin is retained in the cell, and that the conserved s-cyclophilin-specific COOH-terminal extension VEKPFAIAKE is sufficient to direct a secretory protein to s-cyclophilin containing structures. Antibodies to s-cyclophilin-specific peptides were produced and the location of the protein was determined by an immunocytochemical study at the light microscopic level. s-Cyclophilin colocalized with the Ca(2+)-binding protein calreticulin and, to a lesser extent, with the microsomal Ca(2+)-ATPase in the myogenic cell line L6, and with the Ca(2+)-binding protein calsequestrin in skeletal muscle. In activated platelets, s-cyclophilin immunoreactivity was detected in a ring-like structure that might correspond to the Ca(2+)-storing and -releasing dense tubular network. In spreading cells, s-cyclophilin containing vesicular structures accumulated at actin-rich protrusion sites. While s-cyclophilin consistently codistributed with Ca2+ storage site markers, the distribution of s-cyclophilin immunoreactivity was not identical to that of ER markers. To determine whether the COOH-terminal extension of s-cyclophilin was involved in its intracellular transport we added this sequence to the COOH-terminus of the secretory protein glia-derived nexin. Appropriate constructs were expressed transiently in cultured cells and proteins were detected with specific antibodies. We found that glia-derived nexin with the COOH-terminal sequence VEKPFAIAKE (but not with the control sequence GLVVMNIT) colocalized with endogenous s-cyclophilin, indicating that the sequence contained retention information. These results indicate that s-cyclophilin is a retained component of an intracellular organelle and that it may accumulate in specialized portions of the ER, and possibly in calciosomes. Because of its conserved structure, widespread distribution, and abundance s-cyclophilin may be a useful marker to study the biogenesis and distribution of ER subcompartments.
Arber
S
S
Friedrich Miescher Institute, Basel, Switzerland.
Krause
K H
KH
Caroni
P
P
D000591:Amino Acid Isomerases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000032:analysis; D037282:Calreticulin; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002645:Chickens; D016572:Cyclosporine / Q000378:metabolism*; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D019696:Peptidylprolyl Isomerase; D012689:Sequence Homology, Nucleic Acid
D000906:Antibodies; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D002352:Carrier Proteins; D010455:Peptides; D016572:Cyclosporine; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase; D000252:Calcium-Transporting ATPases; D015739:Nocodazole