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

doi:10.1074/jbc.270.22.13084

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 (*)

From the (1) School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588

^§ To whom correspondence and reprint requests should be addressed:
303 Lyman Hall, University of Nebraska, Lincoln, Nebraska 68588-0343
. Tel.: 402-472-1682; Fax: 402-472-8722.

Abstract

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 [S]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.

Footnotes

↵* This work was supported by National Science Foundation Grant MCB-9118721 and funds from the University of Nebraska Center for Biotechnology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ER

endoplasmic reticulum

COS

cytoplasmic osmiophilic structure

LHCPII

light harvesting chlorophyll a/b-binding protein of photosystem II

pLHCPII

precursor to the light harvesting chlorophyll a/b-binding protein of photosystem II

pSSU

precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

SSU

small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

Tricine

N-tris[hydroxymethyl]-methylglycine.
↵²C. Sulli and S. D. Schwartzbach, unpublished observations.