Gram-negative bacteria are surrounded by a complex multilayered cell envelope, consisting of an inner and an outer membrane, and separated by the aqueous periplasm, which contains a thin peptidoglycan cell wall. These bacteria employ an arsenal of highly specialized membrane protein machineries to ensure the correct assembly and maintenance of the membranes forming the cell envelope. Here, we review the diverse protein systems, which perform these functions in Escherichia coli, such as the folding and insertion of membrane proteins, the transport of lipoproteins and lipopolysaccharide within the cell envelope, the targeting of phospholipids, and the regulation of mistargeted envelope components. Some of these protein machineries have been known for a long time, yet still hold surprises. Others have only recently been described and some are still missing pieces or yet remain to be discovered.