The polyprotein precursor to the Euglena light-harvesting chlorophyll a/b-binding protein is transported to the Golgi apparatus prior to chloroplast import and polyprotein processing.
The major Euglena thylakoid protein, the light harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) is synthesized in the cytoplasm as a polyprotein precursor composed of a 141 amino acid presequence containing a signal peptide domain followed by eight mature LHCPIIs covalently linked by a decapeptide. To determine the transport route from cytoplasm to chloroplast and the site of polyprotein processing, Euglena was pulse labeled with [35S]sulfate, organelles separated on sucrose gradients, and pLHCPII and LHCPII immunoprecipitated and separated on SDS gels. After a 10-min pulse, the pLHCPII polyprotein was found in the endoplasmic reticulum (ER) and Golgi apparatus. LHCPII was undetectable after a 10-min pulse consistent with the 20-min half-life for pLHCPII processing. When pulse-labeled cells were chased for 20 or 40 min with unlabeled sulfate, the fraction of pLHCPII in the ER decreased, and the fraction in the Golgi apparatus increased. LHCPII appeared only in thylakoids and chloroplasts, never in the ER or Golgi apparatus. Na2CO3 extraction, a treatment that releases soluble but not integral membrane proteins, did not remove pLHCPII from ER and Golgi membranes. Trypsin digestion of ER and Golgi membranes produced 4 pLHCPII membrane protected fragments. The Euglena pLHCPII polyprotein is transported as an integral membrane protein from the ER to the Golgi apparatus and from the Golgi apparatus to the chloroplast. Polyprotein processing appears to occur during or soon after chloroplast import of the membrane-bound precursor.
Sulli
C
C
School of Biological Sciences, University of Nebraska, Lincoln 68588, USA.
Schwartzbach
S D
SD
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002498:Centrifugation; D002736:Chloroplasts / Q000378:metabolism*; D005056:Euglena gracilis / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013347:Subcellular Fractions / Q000378:metabolism
D002352:Carrier Proteins; D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D011498:Protein Precursors