Identification of ras and ras-related low-molecular-mass GTP-binding proteins associated with rat lung lamellar bodies.
Recent evidence from genetic experiments in yeast and from studies using guanosine triphosphate (GTP) analogues in mammalian cells suggests a key role for low-molecular-mass GTP-binding proteins (LMM-GBPs) (Mr 19 to 28 kD) in processes of intracellular vesicular sorting and secretion. Assembly and exocytosis of the lamellar body (LB), the secretory organelle of the pulmonary alveolar type 2 pneumocyte, may be regulated by LMM-GBPs. We used [alpha-32P]GTP binding to Western blotted proteins, ultraviolet crosslinking of [alpha-32P]GTP to membrane proteins, immunoblotting with specific antisera, and botulinum exoenzyme C3-catalyzed ADP ribosylation to detect LMM-GBPs in LB. With the first two techniques, we have identified six LMM-GBPs of approximately 27, 25.5, 24.5, 23, 22, and 21 kD that are enriched in a highly purified LB fraction compared with type 2 pneumocyte homogenate, crude membranes, and cytosol. Further characterization of the LB LMM-GBPs by immunoblotting revealed that ras p21 is greatly enriched in the LB fraction compared with other type 2 pneumocyte fractions. In addition, botulinum exoenzyme C3 catalyzed the ADP ribosylation of 20- to 21-kD proteins that were similarly enriched in the LB fraction. In contrast, a monospecific antibody to ADP-ribosylation factor reacted with a 19-kD protein only in the type 2 pneumocyte homogenate and cytosol fractions. Monospecific antibodies to yeast Sec4 protein and to rab 3A did not react with any type 2 pneumocyte proteins. The LMM-GBPs specifically associated with LB may participate in intracellular events required for surfactant packaging and secretion.
Rubins
J B
JB
Boston University School of Medicine Pulmonary Center, Massachusetts.
Panchenko
M
M
Shannon
T M
TM
Dickey
B F
BF
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D003176:Complement C3 / Q000378:metabolism; D003432:Cross-Linking Reagents; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D010777:Photochemistry; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D000345:Affinity Labels; D003176:Complement C3; D003432:Cross-Linking Reagents; D010743:Phospholipids; D000246:Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases; D019204:GTP-Binding Proteins; D015689:Oncogene Protein p21(ras)