A yeast-based model system for cloning secreted and membrane proteins.

Marcelo J Surpili,Bernd Müller-Röber,Lothar Willmitzer

doi:10.1590/s0001-37652002000400005

Abstract

The targeting of proteins to cell organelles and membranes, or of proteins destined to secretion, is coordinated by signal sequences located at the 5'-end of their respective genes. A signal sequence trap system was envisaged in which a truncated version of the yeast acid phosphatase pho5 gene lacking the start codon and signal sequence could serve as a reporter gene. A fraction enriched in 5'-end fragments obtained by PCR from a potato guard-cell cDNA library was cloned in frame to the acid phosphatase gene and the acid phosphatase activity was assayed directly in yeast colonies grown on selective medium. Putative signal sequences targeting the acid phosphatase to the membrane or to the outside of the cell were used to screen the cDNA bank in order to recover the original full-size sequence which gave rise to the signal sequence. Two unknown sequences displaying marked tissue-specific expression were retrieved, one of them (YE139) with a higher expression level in green buds and stem cells, and the other one (YE290) with a higher expression level in androceum, gyneceum, and roots. The limitations of the system are further analyzed using other sequences as control.

Highlights

The partitioning of biochemical processes in organelles of eukaryotic cells is one of the major accomplishments of nature
Prior to screening for secreted PHO5 directed by sequences from the cDNA library, the activity of the extracellular acid phosphatase coded by the sequence obtained by PCR with primers PHO1 and PHO2 was tested by fusing a 200 bp sequence containing the probable signal sequence of the sucrose transporter cloned from spinach (Riesmeier et al 1992)
The functionality of the signal sequence trap (SST)-system was demonstrated by inserting the signal sequence from a known membrane protein and later by retrieving two unknown sequences from a guard cell cDNA library showing strong tissue-specific expression

Summary

Introduction

The partitioning of biochemical processes in organelles of eukaryotic cells is one of the major accomplishments of nature. Major targets for the partitioning of proteins in eukaryotes include mitochondria, chloroplasts, peroxisomes, glyoxysomes, and the nucleus Proteins destined to these organelles are translated on free polyribosomes in the cytosol and, concomitantly or shortly thereafter, they are imported into the appropriate organelle based on the Studies on the biosynthesis of secretory proteins in prokaryotes and eukaryotes have shown that most of these proteins are synthesized as precursors containing an additional of 15-30 amino acids at the N-terminal end of the molecule (Milstein et al 1982). The greatest changes in the plasma membrane protein profile occur during the early stages of development, including floral induction and, to a lesser extent, in later stages such as seed formation and senescence These temporal and spatial changes have been studied in detail in highly purified tobacco leaf plasma membrane fractions (Masson and Rossignol 1995)

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