Synaptic vesicles in electromotoneurones. II. Heterogeneity of populations is expressed in uptake properties; exocytosis and insertion of a core proteoglycan into the extracellular matrix.

The three populations of synaptic vesicles in electromotor nerve terminals were analysed quantitatively. Empty vesicles (VP0), fully charged vesicles (VP1) and charged but smaller VP2-type vesicles are present in approximately equal amounts in the nerve terminal. The populations show differences in the kinetics of in vitro uptake of acetylcholine, ATP and Ca2+. VP0 and VP2 accumulate acetylcholine and ATP but no Ca2+, whereas VP1 shows negligible acetylcholine and ATP but high Ca2+ uptake. Thus the expression of uptake properties of this secretory organelle depend on the stage it has reached in its life cycle and might constitute a signal for processing. VP2 was found to contain much less core proteoglycan than VP0 and VP1 indicating that part of it has been lost by exocytosis. In synaptic extracellular matrix containing fractions an antigen is detectable that cross-reacts with an antiserum against the vesicle proteoglycan. This material elutes upon gel filtration in a position similar to a smaller form of proteoglycan found in vesicles. We conclude that the electromotor nerve terminal releases a proteoglycan by the regulated secretory pathway that is deposited in the extracellular matrix. It might have a function in keeping pre- and postsynaptic structures in alignment constituting a transsynaptic signal. Based on the findings described, a model of the vesicles' life-cycle is discussed, whereby the VP2 population is the major source of quantal release of acetylcholine.

Synaptic vesicles in electromotoneurones. I. Axonal transport, site of transmitter uptake and processing of a core proteoglycan during maturation.

We were able by using an in vivo pulse-label technique to trace part of the life cycle of a secretory organelle, the acetylcholine-storing synaptic vesicle from electromotoneurones of Torpedo marmorata. This technique uses [35S]sulphate incorporation into the cell bodies of the electromotoneurones which results in radioactive labelling of a synaptic vesicle heparansulphate proteoglycan--a major core component. Vesicles are anterogradely transported in the axons at a fast rate as 'empty' organelles (VP0 population). In the nerve terminal, maturation of the granule to a population (VP1) fully charged with acetylcholine and ATP occurs. Finally after a longer time interval a change to a third population (VP2) is observed. This population is reduced in diameter as compared to VP0 and VP1 suggesting, in agreement with earlier reports, that it has undergone exo-endocytosis. The changes from VP0 to VP1 and VP2 are accompanied by a degradation of the core proteoglycan as measured by gel filtration of the 35S-labelled compound. The results show that vesicles are axonally transported as preformed organelles, exist in the neurone at least in three different populations and that the nerve terminal is the major site of transmitter uptake.

Nervous system myelin in the electric ray, Torpedo marmorata: Morphological characterization of the membrane and biochemical analysis of its protein components.

In Torpedo, PNS as well as CNS myelines are characterized by clearly separated double intraperiod lines. CNS myelin of Torpedo contains two glycosylated hydrophobic proteins labelled T1 (25,800 Da1) and T2 (29,700 Da1), and two basic proteins BP1 and BP2, migrating like mammalian large basic protein (BP2) and pre-small basic protein (BP1) (Barbarese et al., 1977). PNS myelin of Torpedo carries only BP1 and is characterized by a closely spaced doublet of the glycosylated hydrophobic proteins Con A+ (29,700 Da1) and Con A? (31,000 Da1); the latter does not bind Concanavalin A. These glycosylated proteins (T1, T2, Con A+, Con A?) contain mannose, N-acetylglucosamine and galactose, but lack fucose and sialic acids. They have isoleucine at their amino terminus. They bind anti-rat PNS myelin P(0) antibodies but do not react with anti-rat CNS myelin PLP antibodies. Limited proteolyses of isolated proteins suggest sequence homologies between T1 and T2, and possibly between Con A+ and Con A?. The two basic proteins BP1 and BP2 bind antibodies directed against human myelin basic protein. All Torpedo myelin proteins electrofocus in pH regions characteristic of their mammalian counterparts.